Kinase inhibitors

ABSTRACT

Methods of inhibiting kinases using kinase inhibitors having olefin moieties are disclosed.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §1.119(e) of U.S.Application No. 61/261,696, filed Nov. 16, 2009, and U.S. ApplicationNo. 61/330,271, filed Apr. 30, 2010, each of which is incorporated byreference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under GM071434 awardedby the National Institutes of Health. The Government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

The human genome contains at least 500 genes encoding protein kinases.In fact, protein kinase genes constitute about 2% of all human genes.Protein kinases modify up to 30% of all human proteins and regulate themajority of cellular pathways, particularly those pathways involved insignal transduction.

Because of the profound effects on cells, the activities of proteinkinases are highly regulated. Indeed, unregulated kinase activityfrequently causes disease related to control of cell growth, cellmovement and cell death, particularly cancer. A large body of researchis currently being conducted to find drugs capable of inhibitingspecific kinases to treat a variety of diseases. Some such drugs arealready in clinical use, including Gleevec (imatinib) and Iressa(gefitinib). To increase potency and selectivity, irreversibleelectrophilic inhibitors, which form a covalent bond with a cysteine inthe kinase active site, have been developed. Several of theseirreversible kinase inhibitors are currently in clinical trials (e.g.,neratinib, tovok). Inhibition of proteins through irreversible bindingof an inhibitor to the protein, however, often leads to toxicity and/orimmunogenic problems when used to treat diseases. Therefore, reversiblekinase inhibitors are needed to inhibit kinases while minimizing therisk of toxicity. The present invention addresses these and other needsin the art.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, kinase inhibitors are provided. In some embodiments,the kinase inhibitor has the structure of Formula I:

In Formula I, R¹ is substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

L¹ is bond, —C(O)—, —C(O)N(L³R²)—, —C(O)O—, —S(O)_(n)—, —O—, —N(L³R²)—,—P(O)(OL³R²)O—, —SO₂N(L³R²)—, —P(O)(NL³R²)N—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. The symbol n is 0, 1 or 2.

L² is a bond, —C(O)—, —C(O)N(L^(3A)R^(2A))_(t)—, —C(O)O—, —S(O)_(t)—,—O—, —N(L^(3A)R^(2A))_(t)—, —P(O)(OL^(3A)R^(2A))O—,—SO₂N(L^(3A)R^(2A))—, —P(O)(NL^(3A)R^(2A))N—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. The symbol t is 0, 1 or 2.

L³ and L^(3A) are independently a bond, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. The symbol w is 0, 1 or 2.

R² and R^(2A) are independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

E is an electron withdrawing group or together with L² forms an electronwithdrawing group.

In another aspect, methods of inhibiting protein kinases are provided.The methods include contacting a protein kinase with an effective amountof a kinase inhibitor provided herein. The kinase inhibitor may have thestructure of Formula I or Formula II (or any of the embodiments thereofdescribed herein).

In another aspect, a method of treating a disease associated with kinaseactivity in a subject in need of such treatment. The method includesadministering to the subject an effective amount of a kinase inhibitorprovided herein. The kinase inhibitor may have the structure of FormulaI or Formula II (or any of the embodiments thereof described herein).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B provide mass spectrometric results followingincubation of DMSO or Cmpds 1-4 (FIG. 1A), and Cmpds 5-8 (FIG. 1B) withhuman RSK2 CTD for 1 hr at room temperature.

FIG. 2 depicts recovery of kinase activity of RSK2 CTD after inhibitionby a selection of compounds disclosed herein and subsequent dialysis.Legend: Cmpd 6: checked; Cmpd 7: open box; Cmpd 1: Black box; Cmpd 9:diagonal stripes.

FIG. 3 depicts cyanoacrylate or cyanoacrylamide (Cmpds 4-7) dissociationfrom intact folded RSK2 CTD, as measured by competitive labeling withfluoromethylketone Cmpd 9 (FMK). Upper left panel: time course of FMKlabeling of empty RSK2 CTD. Upper right panel: time course ofdissociation of reversible covalent inhibits, Cmpds 4-7. Lower panel:tabular presentation of dissociation half-time (min) for the indicatedcompounds.

FIG. 4 depicts the formation and reversal of covalent bond formationbetween RSK2 and Cmpd 7 by UV/Visible spectrophotometry. Upper panel:normalized absorbance (400 nm) attributed to Cmpd 7 after reaction witha) C436V RSK2; b) WT RSK2; c) WT RSK2 plus proteinase K; d) WT RSK2 plusSDS; and e) WT RSK2 plus guanidine HCl. Middle panel: UV/Visible spectrafor Cmpd 7, alone in buffer or in the presence of RSK2 or RSK2 plusProteinase K (3 hr incubation). Lower panel: UV/Visible spectra for Cmpd7, alone in buffer or in the presence of RSK2 or RSK2 plus SDS (1 minincubation).

FIG. 5A depicts the mass spectrometric analysis of the incubation ofCmpd 7 with 3 M guanidine HCl. FIG. 5B depicts the mass spectrometricanalysis of the incubation of Cmpd 7 incubated with RSK2 CTD prior toaddition of 3 M guanidine HCl.

FIG. 6 depicts the inhibition of autophosphorylation of Ser386 of RSK2by Cmpds 5-7 and Cmpd 9 in HEK-293 cells. Legend: FMK 9 (Cmpd 9): openbox; Cmpd 7: black box; Cmpd 6: grayed box; Cmpd 5: diagonal stripes.Lower panel: Western blot analysis with phospho-Ser386 RSK2 and anti-HAantibodies, as described herein.

FIG. 7 depicts modes of binding of Cmpds 6, 12 or 15 (top, middle, andlower panels, respectively) to Cys-436 of RSK2, based on X-raycrystallographic structures obtained as described herein.

FIG. 8 depicts modes of binding of Cmpd 40 (top panel) to Cys-436 ofRSK2, and Cmpd 55 to Cys-345 of cSrc.

FIG. 9 depicts ¹H NMR spectrum before and after dilution of reactionmixture.

FIG. 10 depicts cyanoacrylamide absorbance spectrum and graphicalrepresentation of the absorbance values before and after dilution.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e. unbranched) or branched chain,or combination thereof, which may be fully saturated, mono- orpolyunsaturated and can include di- and multivalent radicals, having thenumber of carbon atoms designated (i.e. C₁-C₁₀ means one to tencarbons). Examples of saturated hydrocarbon radicals include, but arenot limited to, groups such as methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs andisomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and thelike. An unsaturated alkyl group is one having one or more double bondsor triple bonds. Examples of unsaturated alkyl groups include, but arenot limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy isan alkyl attached to the remainder of the molecule via an oxygen linker(—O—).

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkyl, as exemplified, but not limited,by —CH₂CH₂CH₂CH₂—, and further includes those groups described below as“heteroalkylene.” Typically, an alkyl (or alkylene) group will have from1 to 24 carbon atoms, with those groups having 10 or fewer carbon atomsbeing preferred in the present invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of atleast one carbon atoms and at least one heteroatom selected from thegroup consisting of O, N, P, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N, P and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—C₁₋₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,—CH═CH—N(CH₃)—CH₃, O—CH₃, —O—CH₂—CH₃, and —CN. Up to two heteroatoms maybe consecutive, such as, for example, —CH₂—NH—OCH₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—. As described above, heteroalkyl groups, as used herein,include those groups that are attached to the remainder of the moleculethrough a heteroatom, such as —C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′,and/or —SO₂R′. Where “heteroalkyl” is recited, followed by recitationsof specific heteroalkyl groups, such as —NR′R″ or the like, it will beunderstood that the terms heteroalkyl and —NR′R″ are not redundant ormutually exclusive. Rather, the specific heteroalkyl groups are recitedto add clarity. Thus, the term “heteroalkyl” should not be interpretedherein as excluding specific heteroalkyl groups, such as —NR′R″ or thelike.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl,and the like. Examples of heterocycloalkyl include, but are not limitedto, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is meant to include, but not be limited to,fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl,4-chlorobutyl, 3-bromopropyl, and the like.

The term “acyl” means —C(O)R where R is a substituted or unsubstitutedalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (preferably from 1 to 3 rings) which are fused together (i.e. afused ring aryl) or linked covalently. A fused ring aryl refers tomultiple rings fused together wherein at least one of the fused rings isan aryl ring. The term “heteroaryl” refers to aryl groups (or rings)that contain from one to four heteroatoms selected from N, O, and S,wherein the nitrogen and sulfur atoms are optionally oxidized, and thenitrogen atom(s) are optionally quaternized. Thus, the term “heteroaryl”includes fused ring heteroaryl groups (i.e. multiple rings fusedtogether wherein at least one of the fused rings is a heteroaromaticring). A 5,6-fused ring heteroarylene refers to two rings fusedtogether, wherein one ring has 5 members and the other ring has 6members, and wherein at least one ring is a heteroaryl ring. Likewise, a6,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 6 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylenerefers to two rings fused together, wherein one ring has 6 members andthe other ring has 5 members, and wherein at least one ring is aheteroaryl ring. A heteroaryl group can be attached to the remainder ofthe molecule through a carbon or heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. An “arylene” and a“heteroarylene,” alone or as part of another substituent means adivalent radical derived from an aryl and heteroaryl, respectively.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

The term “oxo” as used herein means an oxygen that is double bonded to acarbon atom.

The term “alkylsulfonyl” as used herein means a moiety having theformula —S(O₂)—R′, where R′ is an alkyl group as defined above. R′ mayhave a specified number of carbons (e.g. “C₁-C₄ alkylsulfonyl”).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) are meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g.,aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: halogen, —OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl. When a compound of the invention includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″ and R″″ groups when more than one of these groupsis present.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T—C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′— or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(n)—X′—(C″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″ and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl.

As used herein, the term “heteroatom” or “ring heteroatom” is meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

(A)-OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl, substituted with at least one substituent selected from:

(i) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl, substituted with at least one substituent selected from:

(a) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, orheteroaryl, substituted with at least one substituent selected from oxo,—OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₄-C₈cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 4 to 8 membered heterocycloalkyl.

A “lower substituent” or “lower substituent group,” as used herein meansa group selected from all of the substituents described above for a“substituent group,” wherein each substituted or unsubstituted alkyl isa substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and thelike. Also included are salts of amino acids such as arginate and thelike, and salts of organic acids like glucuronic or galactunoric acidsand the like (see, for example, Berge et al., “Pharmaceutical Salts”,Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

Thus, the compounds of the present invention may exist as salts, such aswith pharmaceutically acceptable acids. The present invention includessuch salts. Examples of such salts include hydrochlorides,hydrobromides, sulfates, methanesulfonates, nitrates, maleates,acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates,(−)-tartrates or mixtures thereof including racemic mixtures),succinates, benzoates and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in theart.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,tautomers, geometric isomers and individual isomers are encompassedwithin the scope of the present invention. The compounds of the presentinvention do not include those which are known in the art to be toounstable to synthesize and/or isolate.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areencompassed within the scope of the present invention.

Where a substituent of a compound provided herein is “R-substituted”(e.g. R⁷-substituted), it is meant that the substituent is substitutedwith one or more of the named R groups (e.g. R⁷) as appropriate. In someembodiments, the substituent is substituted with only one of the named Rgroups.

The terms “treating” or “treatment” refers to any indicia of success inthe treatment or amelioration of an injury, pathology or condition,including any objective or subjective parameter such as abatement;remission; diminishing of symptoms or making the injury, pathology orcondition more tolerable to the patient; slowing in the rate ofdegeneration or decline; making the final point of degeneration lessdebilitating; improving a patient's physical or mental well-being. Thetreatment or amelioration of symptoms can be based on objective orsubjective parameters; including the results of a physical examination,neuropsychiatric exams, and/or a psychiatric evaluation. For example,the certain methods presented herein successfully treat cancer bydecreasing the incidence of cancer, in inhibiting its growth and orcausing remission of cancer.

An “effective amount” is an amount of a kinase inhibitor sufficient tocontribute to the treatment, prevention, or reduction of a symptom orsymptoms of a disease, or to inhibit the activity or a protein kinaserelative to the absence of the kinase inhibitor. Where recited inreference to a disease treatment, an “effective amount” may also bereferred to as a “therapeutically effective amount.” A “reduction” of asymptom or symptoms (and grammatical equivalents of this phrase) meansdecreasing of the severity or frequency of the symptom(s), orelimination of the symptom(s). A “prophylactically effective amount” ofa drug is an amount of a drug that, when administered to a subject, willhave the intended prophylactic effect, e.g., preventing or delaying theonset (or reoccurrence) a disease, or reducing the likelihood of theonset (or reoccurrence) of a disease or its symptoms. The fullprophylactic effect does not necessarily occur by administration of onedose, and may occur only after administration of a series of doses.Thus, a prophylactically effective amount may be administered in one ormore administrations. An “activity decreasing amount,” as used herein,refers to an amount of antagonist required to decrease the activity ofan enzyme relative to the absence of the antagonist. A “functiondisrupting amount,” as used herein, refers to the amount of antagonistrequired to disrupt the function of an osteoclast or leukocyte relativeto the absence of the antagonist.

The terms “kinase,” “protein kinase” and the like, refer to an enzymethat transfers a phosphate group from a donor molecule (e.g. ATP) to asubstrate. The process of transferring a phosphate group from a donor toa substrate is conventionally known as phosphorylation.

The term “substrate” in the context of protein phosphorylation refers toa compound (e.g. protein) which accepts a phosphate group and is thusphosphorylated.

II. Kinase Inhibitors

In a first aspect, kinase inhibitors are provided. The kinase inhibitorsare typically reversible kinase inhibitors. In some embodiments, thekinase inhibitor has the structure of Formula I:

In Formula I, R¹ is substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, or-L^(1A)-R^(1A). R^(1A) is substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl. L¹-R¹and/or R¹ is/are generally designed to fit within a kinase ATP bindingsite and/or bind to amino acids within the kinase ATP binding site (e.g.a kinase ATP binding site moiety).

L¹ is a bond, —C(O)—, —C(O)N(L³R²)—, —C(O)O—, —S(O)_(n)—, —O—,—N(L³R²)—, —P(O)(OL³R²)O—, —SO₂N(L³R²)—, —P(O)(NL³R²)N—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. The symbol n is 0, 1 or 2.In some embodiments, L′ is a bond. L^(1A) is a bond, —C(O)—,—C(O)N(L³R^(2′))—, —C(O)O—, —S(O)_(n′)—, —O—, —N(L³R^(2′))—,—P(O)(OL³R^(2′))O—, —SO₂N(L³′R^(2′))—, —P(O)(N L³′R^(2′))N—, substitutedor unsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. The symbol n′ is 0, 1 or 2.

L² is a bond, —C(O)—, —C(O)N(L^(3A)R^(2A))—, —C(O)O—, —S(O)_(t)—, —O—,—N(L^(3A)R^(2A))—, —P(O)(OL^(3A)R^(2A))O—, —SO₂N(L^(3A)R^(2A))—,—P(O)(NL^(3A)R^(2A))N—, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. The symbol t is 0, 1 or 2.

L³, L^(3′) and L^(3A) are independently a bond, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. The symbol w is 0, 1 or 2.

R², R^(2′) and R^(2A) are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

E is an electron withdrawing group or together with L² forms an electronwithdrawing group (e.g. -L²-E may form an electron withdrawing group).In some embodiments, E is ring A or R⁴, wherein R⁴ and ring A are is asdefined below. Thus, E may be hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl or -L^(5A)-R^(4A). E may also be hydrogen,R^(23A)-substituted or unsubstituted alkyl, R^(23A)-substituted orunsubstituted heteroalkyl, R^(23A)-substituted or unsubstitutedcycloalkyl, R^(23A)-substituted or unsubstituted heterocycloalkyl,R^(23A)-substituted or unsubstituted aryl, or R^(23A)-substituted orunsubstituted heteroaryl. In some embodiments, E is a substituted orunsubstituted heteroaryl (e.g. R^(23A)-substituted or unsubstitutedheteroaryl) or substituted or unsubstituted heterocycloalkyl (e.g.R^(23A)-substituted or unsubstituted heterocycloalkyl).

In some embodiments, where -L²-E is an electron withdrawing group, E maysimply be hydrogen. The term “electron withdrawing group” refers to achemical substituent that modifies the electrostatic forces acting on anearby chemical reaction center by withdrawing negative charge from thatchemical reaction center. Thus, electron withdrawing groups drawelectrons away from a reaction center. As a result, the reaction centeris fractionally more positive than it would be in the absence of theelectron-withdrawing group. In some embodiments, the chemical reactioncenter is one of the two carbons forming the carbon-carbon double bond(olefin). In some embodiments, the chemical reaction center is theolefin carbon attached to -L¹-R¹. The electron withdrawing groupfunctions to draw charge or electrons away from this olefin carbonthereby making the olefin carbon electron deficient (relative to theabsence of the electron withdrawing group). The electron deficientolefin carbon is thereby rendered more reactive toward electron richchemical groups, such as the sulfhydryl of a kinase active sitecysteine.

E and -L²-E are typically substituents that sufficiently withdrawelectrons from the reaction center olefin carbon to reversibly bind tothe sulfhydryl of a kinase active cite cysteine (e.g. measurablyreversibly binding when the kinase is fully denatured or partlydenatured). Methods of testing the reversibility of the bond between thereaction center olefin carbon and the sulfhydryl of a kinase active sitecysteine (or a thiol compound proxy) are provided in the Assays andExamples provided below.

In some embodiments, -L²-E is as set forth in one of the Formulae setforth below For example, in Formula II -L²-E is—C(O)X(L¹′—R³)_(z)(L⁵-R⁴), in Formula IIIc -L²-NR³R⁴, in Formula IIIc-L²-E is —WNR³R⁴, etc. Thus, -L²-E may be as set forth in the Formulaeprovided herein and combined with the definitions and embodiments of-L¹-R¹ provided herein. Likewise, -L¹-R¹ may be as set forth in theFormulae provided herein (e.g. Formula IIIa to IIIe wherein -L¹-R¹includes a pyrrolopyrimidinyl) and combined with the definitions of-L²-E as provided herein.

Some non-limiting examples of groups capable of withdrawing electronsfrom a reaction center include, but are not limited to, —NO₂, —N(R₂),—N(R₃)⁺, —N(H₃)⁺, —SO₃H, —SO₃R′, —S(O₂)R′ (sulfone), —S(O)R′(sulfoxide), —S(O₂)NH₂ (sulfonamide), —SO₂NHR′, —SO₂NR′₂, —PO(OR′)₂,—PO₃H₂, —PO(NR′₂)₂, pyridinyl (2-, 3-, 4-), pyrazolyl, indazolyl,imidazolyl, thiazolyl, benzothiazolyl, oxazolyl, benzimidazolyl,benzoxazolyl, isoxazolyl, benzisoxazolyl, triazolyl, benzotriazolyl,quinolinyl, isoquinolinyl, quinazolinyl, pyrimidinyl, a 5 or 6-memberedheteroaryl with a C—N double bond optionally fused to a 5 or 6 memberedheteroaryl, pyridinyl N-oxide, —CX′₃, —C(O)X′, —COOH, —COOR′, —C(O)R′,—C(O)NH₂, —C(O)NHR′, —C(O)NR′₂, —C(O)H, —P(O)(OR′)OR″ and X′, wherein X′is independently halogen (e.g. chloro of fluoro) and R, R′ and R″ areindependently hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, or similarSubstituents (e.g. a substituent group, a size limited substituent groupor a lower substituent group). The term “electron withdrawing group” isdistinguished from an “electron donating group” as known in the art.

Thus, in some embodiments, the kinase inhibitors provided herein (e.g.compounds of Formula I above or the Formulae provided below) arereversible kinase inhibitors, and may measurably dissociate from theprotein kinase when the protein kinase is not denatured, partiallydenatured, or fully denatured. In some embodiments, the covalentreversible kinase inhibitor measurably dissociates from the proteinkinase only when the protein kinase is fully denatured or partiallydenatured, but does not measurably dissociate from the protein kinasewhen the protein kinase is intact, or dissociates at least 10, 1×10²,1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰ fold slower whenthe protein kinase is intact relative to the dissociation when theprotein kinase is fully or partially denatured (referred to herein as a“covalent reversible denatured kinase inhibitor”). In some embodiments,the protein kinase is denatured or fully denatured (i.e. not intact)when placed in denaturing solution, such as 6 N guanidine, 1% SDS, 50%MeCN, or similar protein denaturant, for seconds or minutes (e.g. 30 to120 seconds, such as 60 seconds). In some embodiments, the reversiblekinase inhibitors described herein, after covalently binding to thekinase active site cysteine residue, are capable of dissociating fromthe kinase within seconds or minutes after denaturing/unfolding thekinase with 6 N guanidine, 1% SDS, 50% MeCN, or similar proteindenaturant.

In some embodiments of the kinase inhibitors provided herein, if L′ is abond and R′ is(3-(4-amino-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)propan-1-ol)-6-yl,then -L²-E is not —C(O)NH₂. In other embodiments, where L¹ is a bond andR¹ is a substituted 4-amino pyrrolopyrimidinyl, then -L²-E is not—C(O)NH₂. In other embodiments , where L′ is a bond and R¹ is asubstituted pyrrolopyrimidinyl, then -L²-E is not —C(O)NH₂. In certainembodiments, where L¹ is a bond and R¹ is a substituted or unsubstitutedpyrrolopyrimidinyl, then -L²-E is not —C(O)NH₂. In other embodiments,where R¹ is a substituted or unsubstituted pyrrolopyrimidinyl, then-L²-E is not —C(O)NH₂.

In other embodiments, -L²-E is not —C(O)NH₂. In other embodiments, -L²-Eis not —C(O)OH, or —C(O)OR″, wherein R″ is an unsubstituted C₁-C₁₀ alkyl(e.g. unsubstituted C₁—O₅ alkyl such as methyl). In some embodiments,-L²-E is not —C(O)N(CH₃)₂ or —C(O)NH(CH₃)

In some embodiments of Formula I above or the Formulae provided below,R′ and R^(1A) are independently R⁷-substituted or unsubstituted alkyl,R⁷-substituted or unsubstituted heteroalkyl, R⁷-substituted orunsubstituted cycloalkyl, R⁷-substituted or unsubstitutedheterocycloalkyl, R⁷-substituted or unsubstituted aryl, orR⁷-substituted or unsubstituted heteroaryl.

R⁷ is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R⁸-substituted or unsubstituted alkyl, R⁸-substituted or unsubstitutedheteroalkyl, R⁸-substituted or unsubstituted cycloalkyl, R⁸-substitutedor unsubstituted heterocycloalkyl, R⁸-substituted or unsubstituted aryl,R⁸-substituted or unsubstituted heteroaryl, or -L⁶-R^(7A). L⁶ is —O—,—NH—, —C(O)—, —C(O)NH—, —S(O)_(m)—, or —S(O)_(m)NH—, where m is 0, 1, or2.

R^(7A) is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R⁸-substituted or unsubstituted alkyl, R⁸-substituted or unsubstitutedheteroalkyl, R⁸-substituted or unsubstituted cycloalkyl, R⁸-substitutedor unsubstituted heterocycloalkyl, R⁸-substituted or unsubstituted aryl,or R⁸-substituted or unsubstituted heteroaryl. R⁸ is independentlyhydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃, R⁹-substituted orunsubstituted alkyl, R⁹-substituted or unsubstituted heteroalkyl,R⁹-substituted or unsubstituted cycloalkyl, R⁹-substituted orunsubstituted heterocycloalkyl, R⁹-substituted or unsubstituted aryl,R⁹-substituted or unsubstituted heteroaryl or -L⁹-R^(9A). In someembodiments, R⁸ is independently —OH or unsubstituted alkyl. L⁹ is —O—,—NH—, —C(O)—, —C(O)NH—, —S(O)_(m)′—, or —S(O)_(m′)NH—, where m′ is 0, 1,or 2.

R^(9A) is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R¹⁰-substituted or unsubstituted alkyl, R¹⁰-substituted or unsubstitutedheteroalkyl, R¹⁰-substituted or unsubstituted cycloalkyl,R¹⁰-substituted or unsubstituted heterocycloalkyl, R¹⁰-substituted orunsubstituted aryl, or R¹⁰-substituted or unsubstituted heteroaryl. R⁹is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R¹⁰-substituted or unsubstituted alkyl, R¹⁰-substituted or unsubstitutedheteroalkyl, R¹⁰-substituted or unsubstituted cycloalkyl,R¹⁰-substituted or unsubstituted heterocycloalkyl, R¹⁰-substituted orunsubstituted aryl, or R¹⁰-substituted or unsubstituted heteroaryl. R¹⁰is independently halogen, —CN, —OH, —NH₂, —COOH, —CF₃, unsubstitutedalkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstitutedheteroaryl.

In some embodiments, R¹ and R^(1A) are independently R⁷-substituted orunsubstituted 6,5 fused ring heteroaryl, R⁷-substituted or unsubstituted5,6 fused ring heteroaryl, R⁷-substituted or unsubstituted 5,5 fusedring heteroaryl, R⁷-substituted or unsubstituted 6,6 fused ringheteroaryl, or R⁷-substituted or unsubstituted 5 or 6 memberedheteroaryl having at least 2 (e.g. 2 to 4) ring nitrogens. In certainembodiments, R¹ and R^(1A) are independently R⁷-substituted phenyl,R⁷-substituted piperidinyl, R⁷-substituted 6-membered heterocycloalkyl,R⁷-substituted or unsubstituted 6,5 fused ring heteroaryl,R⁷-substituted or unsubstituted 5,6 fused ring heteroaryl. R⁷ may behalogen, —CN, —OH, —COOH, R⁸-substituted or unsubstituted alkyl,R⁸-substituted or unsubstituted heteroalkyl, R⁸-substituted orunsubstituted cycloalkyl, R⁸-substituted or unsubstitutedheterocycloalkyl, R⁸-substituted or unsubstituted aryl, orR⁸-substituted or unsubstituted heteroaryl or -L⁶-R^(7A). R^(7A) may beR⁸-substituted or unsubstituted cycloalkyl, R⁸-substituted orunsubstituted heterocycloalkyl, R⁸-substituted or unsubstituted aryl, orR⁸-substituted or unsubstituted heteroaryl. L⁶ may be —O—, —NH—, —C(O)—,—C(O)NH—, —S(O)_(m)—, or —S(O)_(m)NH—. R⁸ may be —OH, or R⁹-substitutedor unsubstituted alkyl. In some related embodiments, R⁷ is independentlyR⁸-substituted or unsubstituted cycloalkyl, R⁸-substituted orunsubstituted heterocycloalkyl, R⁸-substituted or unsubstituted aryl,R⁸-substituted or unsubstituted heteroaryl, or -L⁶-R^(7A). In otherrelated embodiments, R⁷ is R⁸-substituted or unsubstituted heteroaryl,or -L⁶-R^(7A). L⁶ may be —C(O)—. R^(7A) may be R⁸-substituted orunsubstituted heteroaryl.

In some embodiments, R¹ or R^(1A) is a substituted or unsubstitutedheteroaryl, such as an R⁷-substituted or unsubstituted heteroaryl. Theheteroaryl may be a substituted or unsubstituted pyrrolopyrimidinyl,substituted or unsubstituted indolyl, substituted or unsubstitutedpyrazolyl, substituted or unsubstituted indazolyl, substituted orunsubstituted imidazolyl, substituted or unsubstituted thiazolyl,substituted or unsubstituted benzothiazolyl, substituted orunsubstituted oxazolyl, substituted or unsubstituted benzimidazolyl,substituted or unsubstituted benzoxazolyl, substituted or unsubstitutedisoxazolyl, substituted or unsubstituted benzisoxazolyl, substituted orunsubstituted triazolyl, substituted or unsubstituted benzotriazolyl,substituted or unsubstituted quinolinyl, substituted or unsubstitutedisoquinolinyl, substituted or unsubstituted quinazolinyl, substituted orunsubstituted pyrimidinyl, substituted or unsubstituted pyridinylN-oxide, substituted or unsubstituted furanyl, substituted orunsubstituted thiophenyl, substituted or unsubstituted benzofuranyl,substituted or unsubstituted benzothiophenyl, substituted orunsubstituted imidazo[1,2b]pyridazinyl. In some embodiments, R¹ orR^(1A) is a substituted or unsubstituted 6,5 fused ring heteroaryl, asubstituted or unsubstituted 5,6 fused ring heteroaryl, a substituted orunsubstituted 5,5 fused ring heteroaryl, or a substituted orunsubstituted 6,6 fused ring heteroaryl. In other embodiments, R¹ orR^(1A) is a substituted or unsubstituted 5 or 6 membered heteroarylhaving at least 2 (e.g. 2 to 4) ring nitrogens. As discussed above anyR¹ substituent may be R⁷-substituted, including the substituents recitedin this paragraph.

R¹ and/or -L¹-R¹ is/are generally designed to be a kinase ATP bindingsite moiety. It has also been found herein that compounds of Formula Iin which R¹ and/or -L¹-R¹ is/are attached to the remainder of thecompound via an sp2 carbon, stability of the compound is improved. A“kinase ATP binding site moiety,” as used herein, is a moiety capable offitting within a kinase ATP binding site and/or binding to amino acidswithin the kinase ATP binding site. Kinase ATP binding sites are wellknown for wide variety of kinases, and may be easily determined from theprimary amino acid structure of a kinase using computer modelingtechniques commonly employed in the art. In certain embodiments, -L¹-R¹is a kinase ATP binding site moiety and the electron deficient olefincarbon binds to a sulfhydryl of a kinase active site cysteine. Thus, insome embodiments the kinase inhibitors provided herein bind to at leasttwo points of the protein kinase: at least one residue within the ATPbinding site moiety and a sulfhydryl of a kinase active site cysteine.In some embodiments, -L¹-R¹ does not have the formula:

In other embodiments, -L¹-R¹ is not a phenyl substituted with hydroxyl.In some embodiments, -L¹-R¹ includes a substituted or unsubstitutedheteroaryl or substituted or unsubstituted heteroarylene group.

In some embodiments, L¹-R¹ and/or R¹ is substituted or unsubstitutedaryl or substituted or unsubstituted heteroaryl. In other embodiments,L¹ is a bond and R¹ is substituted or unsubstituted aryl or substitutedor unsubstituted heteroaryl. In other embodiments, L¹ is a substitutedor unsubstituted arylene or substituted or unsubstituted heteroaryleneand R¹ or R^(1A) is a substituted or unsubstituted aryl or substitutedor unsubstituted heteroaryl. For example, L¹ may be substituted orunsubstituted arylene and R¹ or R^(1A) may be substituted orunsubstituted heteroaryl. In related embodiments, L^(1A) is —C(O)—,—C(O)NH—, —C(O)O—, —S(O)—, —SO₂—. —S—, —O—, —NH— or —SO₂NH—.

In some embodiments, L¹ is a bond and R¹ is an R⁷-substituted phenyl. Insome related embodiments, R⁷ is -L⁶-R^(7A) or R⁸-substituted orunsubstituted heteroaryl, wherein R^(7A) is R⁸-substituted orunsubstituted heteroaryl. In some further related embodiments, L⁶ is abond or —C(O)—. In other related embodiment, the R⁸-substituted orunsubstituted heteroaryl is an R⁸-substituted or unsubstituted 6,5 fusedring heteroaryl or R⁸-substituted or unsubstituted 5,6 fused ring.

In other embodiments, L¹ is a bond and R¹ is an R⁷-substituted phenyl,R⁷-substituted piperidinyl, R⁷-substituted piperizinyl, R⁷-substitutedpyrrolidinyl, R⁷-substituted piperidinyl, R⁷-substituted azepanyl, orR⁷-substituted azetidinyl. In some related embodiments, R⁷ is -L⁶-R^(7A)or R⁸-substituted or unsubstituted heteroaryl, wherein R^(7A) isR⁸-substituted or unsubstituted heteroaryl. In some further relatedembodiments, L⁶ is a bond or —C(O)—. In other related embodiment, theR⁸-substituted or unsubstituted heteroaryl is an R⁸-substituted orunsubstituted 6,5 fused ring heteroaryl or R⁸-substituted orunsubstituted 5,6 fused ring.

In some embodiment, L′ is a substituted or unsubstituted arylene (e.g.phenylene) and R¹ or R^(1A) is a substituted or unsubstituted heteroaryl(e.g. R⁷-substituted). In other embodiments, L′ is a substituted orunsubstituted heteroarylene.

In some embodiments of Formula I above or the Formulae provided below,L′ is a bond, —C(O)—, —C(O)N(L³R²)—, —C(O)O—, —S(O)_(n)—, —O—, —S—,—N(L³R²)—, —P(O)(OL³R²)O—, —SO₂N(L³R²)—, —P(O)(NL³R²)N—, R¹¹-substitutedor unsubstituted alkylene, R¹¹-substituted or unsubstitutedheteroalkylene, R¹¹-substituted or unsubstituted cycloalkylene,R¹¹-substituted or unsubstituted heterocycloalkylene, R¹¹-substituted orunsubstituted arylene, or R¹¹-substituted or unsubstitutedheteroarylene. L^(1A) may be a bond, —C(O)—, —C(O)N(L³R^(2′))-, —C(O)O—,—S(O)_(n′)—, —O—, —N(L³R^(2′))-, —P(O)(OL³R^(2′))O—, —SO₂N(L³R^(2′))-,—P(O)(N L³R^(2′))N—, R¹¹-substituted or unsubstituted alkylene,R¹¹-substituted or unsubstituted heteroalkylene, R¹¹-substituted orunsubstituted cycloalkylene, R¹¹-substituted or unsubstitutedheterocycloalkylene, R¹¹-substituted or unsubstituted arylene, orR¹¹-substituted or unsubstituted heteroarylene.

L¹ and L^(1A) may also independently be a bond, R¹¹-substituted orunsubstituted alkylene, R¹¹-substituted or unsubstituted heteroalkylene,R¹¹-substituted or unsubstituted cycloalkylene, R¹¹-substituted orunsubstituted heterocycloalkylene, R¹¹-substituted or unsubstitutedarylene, or R¹¹-substituted or unsubstituted heteroarylene.

R¹¹ is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R¹²-substituted or unsubstituted alkyl, R¹²-substituted or unsubstitutedheteroalkyl, R¹²-substituted or unsubstituted cycloalkyl,R¹²-substituted or unsubstituted heterocycloalkyl, R¹²-substituted orunsubstituted aryl, or R¹²-substituted or unsubstituted heteroaryl. R¹²is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R¹³-substituted or unsubstituted alkyl, R¹³-substituted or unsubstitutedheteroalkyl, R¹³-substituted or unsubstituted cycloalkyl,R¹³-substituted or unsubstituted heterocycloalkyl, R¹³-substituted orunsubstituted aryl, or R¹³-substituted or unsubstituted heteroaryl. R¹³is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R¹⁴-substituted or unsubstituted alkyl, R¹⁴-substituted or unsubstitutedheteroalkyl, R¹⁴-substituted or unsubstituted cycloalkyl,R¹⁴-substituted or unsubstituted heterocycloalkyl, R¹⁴-substituted orunsubstituted aryl, or R¹⁴-substituted or unsubstituted heteroaryl. R¹⁴is independently halogen, —CN, —OH, —NH₂, —COOH, —CF₃, unsubstitutedalkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstitutedheteroaryl.

L² may be a bond, —C(O)N(L^(3A)R^(2A))—, —C(O)O—, —S(O)_(t)—, —O—, —S—,—N(L^(3A)R^(2A))—, —C(O)—P(O)(OL³R²))-, —SO₂N(L³R²)—, —P(O)(NL³R²)N—,R¹⁹-substituted or unsubstituted alkylene, R¹⁹-substituted orunsubstituted heteroalkylene, R¹⁹-substituted or unsubstitutedcycloalkylene, R¹⁹-substituted or unsubstituted heterocycloalkylene,R¹⁹-substituted or unsubstituted arylene, or R¹⁹-substituted orunsubstituted heteroarylene.

R¹⁹ is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R^(N)-substituted or unsubstituted alkyl, R^(N)-substituted orunsubstituted heteroalkyl, R²⁰-substituted or unsubstituted cycloalkyl,R²⁰-substituted or unsubstituted heterocycloalkyl, R²⁰-substituted orunsubstituted aryl, or R²⁰-substituted or unsubstituted heteroaryl. R²⁰is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R²¹-substituted or unsubstituted alkyl, R^(2′)-substituted orunsubstituted heteroalkyl, R²¹-substituted or unsubstituted cycloalkyl,R²¹-substituted or unsubstituted heterocycloalkyl, R²¹-substituted orunsubstituted aryl, or R²¹-substituted or unsubstituted heteroaryl. R²¹is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R²²-substituted or unsubstituted alkyl, R²²-substituted or unsubstitutedheteroalkyl, R²²-substituted or unsubstituted cycloalkyl,R²²-substituted or unsubstituted heterocycloalkyl, R²²-substituted orunsubstituted aryl, or R²²-substituted or unsubstituted heteroaryl. R²²is independently halogen, —CN, —OH, —NH₂, —COOH, —CF₃, unsubstitutedalkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstitutedheteroaryl.

In some embodiments, L³, L^(3′), and L^(3A) are independently a bond,R²⁷-substituted or unsubstituted alkylene, R²⁷-substituted orunsubstituted heteroalkylene, R²⁷-substituted or unsubstitutedcycloalkylene, R²⁷-substituted or unsubstituted heterocycloalkylene,R²⁷-substituted or unsubstituted arylene, or R²⁷-substituted orunsubstituted heteroarylene. R²⁷ is independently hydrogen, halogen,—CN, —OH, —NH₂, —COOH, —CF₃, R²⁸-substituted or unsubstituted alkyl,R²⁸-substituted or unsubstituted heteroalkyl, R²⁸-substituted orunsubstituted cycloalkyl, R²⁸-substituted or unsubstitutedheterocycloalkyl, R²⁸-substituted or unsubstituted aryl, orR²⁸-substituted or unsubstituted heteroaryl. R²⁸ is independentlyhydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃, R²⁹-substituted orunsubstituted alkyl, R²⁹-substituted or unsubstituted heteroalkyl,R²⁹-substituted or unsubstituted cycloalkyl, R²⁹-substituted orunsubstituted heterocycloalkyl, R²⁹-substituted or unsubstituted aryl,or R²⁹-substituted or unsubstituted heteroaryl. R²⁹ is independentlyhydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃, R^(N)-substituted orunsubstituted alkyl, R^(N)-substituted or unsubstituted heteroalkyl,R³⁰-substituted or unsubstituted cycloalkyl, R³⁰-substituted orunsubstituted heterocycloalkyl, R³⁰-substituted or unsubstituted aryl,or R³⁰-substituted or unsubstituted heteroaryl. R³⁰ is independentlyhalogen, —CN, —OH, —NH₂, —COOH, —CF₃, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl.

In some embodiments, R², R^(2′), and R^(2A) are independently hydrogen,R¹⁵-substituted or unsubstituted alkyl, R¹⁵-substituted or unsubstitutedheteroalkyl, R¹⁵-substituted or unsubstituted cycloalkyl,R¹⁵-substituted or unsubstituted heterocycloalkyl, R¹⁵-substituted orunsubstituted aryl, or R¹⁵-substituted or unsubstituted heteroaryl.

R¹⁵ is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R¹⁶-substituted or unsubstituted alkyl, R¹⁶-substituted or unsubstitutedheteroalkyl, R¹⁶-substituted or unsubstituted cycloalkyl,R¹⁶-substituted or unsubstituted heterocycloalkyl, R¹⁶-substituted orunsubstituted aryl, R¹⁶-substituted or unsubstituted heteroaryl, or-L⁷-R^(15A). L⁷ is independently —O—, —C(O)—, —C(O)NH—, —S(O)_(Y)—, or—S(O)_(y)NH—, where y is 0, 1, or 2. R^(15A) is independently hydrogen,halogen, —CN, —OH, —NH₂, —COOH, —CF₃, R¹⁶-substituted or unsubstitutedalkyl, R¹⁶-substituted or unsubstituted heteroalkyl, R¹⁶-substituted orunsubstituted cycloalkyl, R¹⁶-substituted or unsubstitutedheterocycloalkyl, R¹⁶-substituted or unsubstituted aryl, R¹⁶-substitutedor unsubstituted heteroaryl. R¹⁶ is independently hydrogen, halogen,—CN, —OH, —NH₂, —COOH, —CF₃, R¹⁷-substituted or unsubstituted alkyl,R¹⁷-substituted or unsubstituted heteroalkyl, R¹⁷-substituted orunsubstituted cycloalkyl, R¹⁷-substituted or unsubstitutedheterocycloalkyl, R¹⁷-substituted or unsubstituted aryl, orR¹⁷-substituted or unsubstituted heteroaryl. R¹⁷ is independentlyhydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃, R¹⁸-substituted orunsubstituted alkyl, R¹⁸-substituted or unsubstituted heteroalkyl,R¹⁸-substituted or unsubstituted cycloalkyl, R¹⁸-substituted orunsubstituted heterocycloalkyl, R¹⁸-substituted or unsubstituted aryl,or R¹⁸-substituted or unsubstituted heteroaryl. R¹⁸ is independentlyhalogen, —CN, —OH, —NH₂, —COOH, —CF₃, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl.

In some embodiments, the kinase inhibitor has the structure of FormulaII:

Regarding Formula II, W is —C(O)— or —S(O)₂—, X is O or N, and z is 0 or1, provided, however, that if X is O, then z is 0. L¹ and R¹ are definedas disclosed above for Formula I. In some embodiments, X is N. In otherembodiments, X is O.

R³ is hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

R⁴ is hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl or-L^(5A)-R^(4A). R^(4A) is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl. R³ andR⁴ may be joined together with X to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl. R³ andR^(4A) may be joined together with X to form a substituted orunsubstituted heterocycloalkyl or substituted or unsubstitutedheteroaryl.

L⁴ and L⁵ are independently a bond, —C(O)—, —C(O)N(L^(3A)R^(2A))—,—C(O)O—, —S(O)_(t), —O—, —N(L^(3A)R^(2A))—, —P(O)(OL^(3A)R^(2A))O—,—SO₂N(L^(3A)R^(2A))—, —P(O)(NL^(3A)R^(2A))N—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. L^(3A), R^(2A), t are asdefined above. L^(5A) is a bond, —C(O)—, —C(O)N(L^(3A′)R^(2A′))—,—C(O)O—, —S(O)_(t′)—, —O—, —N(L^(3A′)R^(2A′))—, —P(O)(OL^(3A′)R^(2A′))O—, —SO₂N(L^(3A′)R^(2A′))—, —P(O)(NL^(3A′)R^(2A′))N—,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene. The symbol t′ is0, 1 or 2.

R^(2A′) is independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. Insome embodiments, R^(2′) is independently hydrogen, R¹⁵-substituted orunsubstituted alkyl, R¹⁵-substituted or unsubstituted heteroalkyl,R¹⁵-substituted or unsubstituted cycloalkyl, R¹⁵-substituted orunsubstituted heterocycloalkyl, R¹⁵-substituted or unsubstituted aryl,or R¹⁵-substituted or unsubstituted heteroaryl. R¹⁵ is as defined above.

L^(3A′) is independently a bond, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. In some embodiments, L^(3A′)is a bond, R²⁷-substituted or unsubstituted alkylene, R²⁷-substituted orunsubstituted heteroalkylene, R²⁷-substituted or unsubstitutedcycloalkylene, R²⁷-substituted or unsubstituted heterocycloalkylene,R²⁷-substituted or unsubstituted arylene, or R²⁷-substituted orunsubstituted heteroarylene. R²⁷ is as defined above.

In some embodiments, L⁴ and L⁵ are independently a bond, —C(O)—,—C(O)N(L^(3A)R^(2A))—, —C(O)O—, —S(O)_(t)—, —O—, —N(L^(3A)R^(2A))—,—P(O)(OL^(3A)R^(2A))O—, —SO₂N(L^(3A)R^(2A))—, —P(O)(NL^(3A)R^(2A))N—,R¹⁹-substituted or unsubstituted alkylene, R¹⁹-substituted orunsubstituted heteroalkylene, R¹⁹-substituted or unsubstitutedcycloalkylene, R¹⁹-substituted or unsubstituted heterocycloalkylene,R¹⁹-substituted or unsubstituted arylene, or R¹⁹-substituted orunsubstituted heteroarylene. In some embodiments, L^(5A) is a bond,—C(O)—, —C(O)N(L^(3A′)R^(2A′))C(O)O—, —S(O)_(t′)—, —O—,—N(L^(3A′)R^(2′))P(O)(O L^(3A′)R^(2A′))O—, —SO₂N(L^(3A′)R^(2A′))—,—P(O)(NL^(3A′)R^(2A′))N—, R¹⁹-substituted or unsubstituted alkylene,R¹⁹-substituted or unsubstituted heteroalkylene, R¹⁹-substituted orunsubstituted cycloalkylene, R¹⁹-substituted or unsubstitutedheterocycloalkylene, R¹⁹-substituted or unsubstituted arylene, orR¹⁹-substituted or unsubstituted heteroarylene. R¹⁹ is as defined above.

In some embodiments, at least one of L⁵-R⁴ or L⁴-R³ includes asubstituted or unsubstituted heteroaryl or substituted or unsubstitutedheteroarylene. In some embodiments, one of L⁵-R⁴ or L⁴-R³ includes asubstituted or unsubstituted heteroaryl or substituted or unsubstitutedheteroarylene. In some embodiments, one of L⁵-R⁴ or L⁴-R³ is a hydrogen.

In some embodiments, if L¹ is a bond and R¹ is(3-(4-amino-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)propan-1-ol)-6-yl,then at least one of R³ and R⁴ are not hydrogen.

In some embodiments of Formula I or Formula II, R¹ or R^(1A) isR⁷-substituted or unsubstituted heterocycloalkyl, R⁷-substituted orunsubstituted aryl, or R⁷-substituted or unsubstituted heteroaryl,wherein R⁷ is as described above.

In some embodiments, R³ is hydrogen, R²³-substituted or unsubstitutedalkyl, R²³-substituted or unsubstituted heteroalkyl, R²³-substituted orunsubstituted cycloalkyl, R²³-substituted or unsubstitutedheterocycloalkyl, R²³-substituted or unsubstituted aryl, orR²³-substituted or unsubstituted heteroaryl.

R²³ is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R²⁴-substituted or unsubstituted alkyl, R²⁴-substituted or unsubstitutedheteroalkyl, R²⁴-substituted or unsubstituted cycloalkyl,R²⁴-substituted or unsubstituted heterocycloalkyl, R²⁴-substituted orunsubstituted aryl, R²⁴-substituted or unsubstituted heteroaryl, or-L⁸-R^(23A′). -L⁸ is independently —O—, —C(O)—, —C(O)NH—, —S(O)_(p)—, or—S(O)_(p)NH—, wherein p is 0, I, or 2. R^(23A′) is independentlyhydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃, R²⁴-substituted orunsubstituted alkyl, R²⁴-substituted or unsubstituted heteroalkyl,R²⁴-substituted or unsubstituted cycloalkyl, R²⁴-substituted orunsubstituted heterocycloalkyl, R²⁴-substituted or unsubstituted aryl,R²⁴-substituted or unsubstituted heteroaryl. R²⁴ is independentlyhydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃, R²⁵-substituted orunsubstituted alkyl, R²⁵-substituted or unsubstituted heteroalkyl,R²⁵-substituted or unsubstituted cycloalkyl, R²⁵-substituted orunsubstituted heterocycloalkyl, R²⁵-substituted or unsubstituted aryl,or R²⁵-substituted or unsubstituted heteroaryl. R²⁵ is independentlyhydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃, R²⁶-substituted orunsubstituted alkyl, R²⁶-substituted or unsubstituted heteroalkyl,R²⁶-substituted or unsubstituted cycloalkyl, R²⁶-substituted orunsubstituted heterocycloalkyl, R²⁶-substituted or unsubstituted aryl,or R²⁶-substituted or unsubstituted heteroaryl. R²⁶ is independentlyhalogen, —CN, —OH, —NH₂, —COOH, —CF₃, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl.

In some embodiments, R³ and R⁴ are joined together with X to form asubstituted or unsubstituted heteroaryl or substituted or unsubstitutedheterocycloalkyl (e.g. R²³-substituted or unsubstituted heteroaryl orR²³-substituted or unsubstituted heterocycloalkyl). In some embodiments,R³ and R⁴ are joined together with X to form a 4 to 8 memberedsubstituted or unsubstituted heterocycloalkyl or a 5 to 6 memberedsubstituted or unsubstituted heteroaryl (e.g. R²³-substituted speciesthereof). In some embodiments, R³ and R⁴ are joined together with X toform a 4 to 7 membered substituted or unsubstituted heterocycloalkyl ora 5 to 6 membered substituted or unsubstituted heteroaryl (e.g.R²³-substituted species thereof). In some embodiments, R³ and R⁴ arejoined together with X to form a 5 to 7 membered substituted orunsubstituted heterocycloalkyl or a 5 to 6 membered substituted orunsubstituted heteroaryl (e.g. R²³-substituted species thereof). In someembodiments, R³ and R⁴ are joined together with X to form a substitutedor unsubstituted morpholino, substituted or unsubstituted thiomorpholino(or oxidated ring thereof), substituted or unsubstituted pyridinyl,substituted or unsubstituted pyrazyinyl, substituted or unsubstitutedpyrrolidinyl, substituted or unsubstituted piperidinyl, substituted orunsubstituted piperizinyl, substituted or unsubstituted pyrrolidinyl,substituted or unsubstituted azepanyl, or substituted or unsubstitutedazetidinyl (e.g. R²³-substituted substituents thereof).

In some embodiments relating to Formula II, R⁴ and R^(4A) are hydrogen,R^(23A)-substituted or unsubstituted alkyl, R^(23A)-substituted orunsubstituted heteroalkyl, R^(23A)-substituted or unsubstitutedcycloalkyl, R^(23A)-substituted or unsubstituted heterocycloalkyl,R^(23A)-substituted or unsubstituted aryl, or R^(23A)-substituted orunsubstituted heteroaryl. In some embodiments, R⁴ and R^(4A) are nothydrogen.

R^(23A) is hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R^(24A)-substituted or unsubstituted alkyl, R^(24A)-substituted orunsubstituted heteroalkyl, R^(24A)-substituted or unsubstitutedcycloalkyl, R^(24A)-substituted or unsubstituted heterocycloalkyl,R^(24A)-substituted or unsubstituted aryl, R^(24A)-substituted orunsubstituted heteroaryl, or -L^(7A)-R^(24B). L^(7A) is independently—O—, —C(O)—, —C(O)NH—, —S(O)_(y′)—, or —S(O)_(y)NH—, wherein y′ is 0, 1,or 2. R^(24B) is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH,—CF₃, R^(24A)-substituted or unsubstituted alkyl, R^(24A)-substituted orunsubstituted heteroalkyl, R^(24A)-substituted or unsubstitutedcycloalkyl, R^(24A)-substituted or unsubstituted heterocycloalkyl,R^(24A)-substituted or unsubstituted aryl, R^(24A)-substituted orunsubstituted heteroaryl. R^(24A) is independently hydrogen, halogen,—CN, —OH, —NH₂, —COOH, —CF₃, R^(25A)-substituted or unsubstituted alkyl,R^(25A)-substituted or unsubstituted heteroalkyl, R^(25A)-substituted orunsubstituted cycloalkyl, R^(25A)-substituted or unsubstitutedheterocycloalkyl, R^(25A)-substituted or unsubstituted aryl, orR^(25A)-substituted or unsubstituted heteroaryl. R^(25A) isindependently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R^(26A)-substituted or unsubstituted alkyl, R^(26A)-substituted orunsubstituted heteroalkyl, R^(26A)-substituted or unsubstitutedcycloalkyl, R^(26A)-substituted or unsubstituted heterocycloalkyl,R^(26A)-substituted or unsubstituted aryl, or R^(26A)-substituted orunsubstituted heteroaryl. R^(26A) is independently halogen, —CN, —OH,—NH₂, —COON, —CF₃, unsubstituted alkyl, unsubstituted heteroalkyl,unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstitutedaryl, or unsubstituted heteroaryl.

In some embodiments, R⁴ or R^(4A) is R^(23A)-substituted orunsubstituted alkyl. In some embodiments, R³ is substituted orunsubstituted pyridinyl (2-, 3-, 4-), substituted or unsubstitutedpyrazolyl, substituted or unsubstituted indazolyl, substituted orunsubstituted imidazolyl, substituted or unsubstituted thiazolyl,substituted or unsubstituted benzothiazolyl, substituted orunsubstituted oxazolyl, substituted or unsubstituted benzimidazolyl,substituted or unsubstituted benzoxazolyl, substituted or unsubstitutedisoxazolyl, substituted or unsubstituted benzisoxazolyl, substituted orunsubstituted triazolyl, substituted or unsubstituted benzotriazolyl,substituted or unsubstituted quinolinyl, substituted or unsubstitutedisoquinolinyl, substituted or unsubstituted quinazolinyl, substituted orunsubstituted pyrimidinyl, substituted or unsubstituted pyridinylN-oxide, or a substituted or unsubstituted 5 or 6-membered heteroarylwith at least one ring nitrogen (e.g. a C—N double bond), optionallyfused to a 5 or 6 membered heteroaryl. In some embodiments where thesubstituents in the preceding sentence are substituted, the substituentis R¹⁵-substituted.

Further regarding compounds with the structure of Formula II, in someembodiments L¹, L⁴ and L⁵ are independently a bond, and R¹ isR⁷-substituted or unsubstituted heterocycloalkyl, R⁷-substituted orunsubstituted aryl, or R⁷-substituted or unsubstituted heteroaryl. Insome embodiments, R⁷ is independently —NH₂, R⁸-substituted orunsubstituted alkyl, R⁸-substituted or unsubstituted aryl,R⁸-substituted or unsubstituted heteroaryl, or -L⁶-R^(7A). L⁶ may be—C(O)—. R^(7A) may be R⁸-substituted or unsubstituted alkyl,R⁸-substituted or unsubstituted aryl, R⁸-substituted or unsubstitutedheteroaryl. R⁸ may be —OH or R⁹-substituted or unsubstituted alkyl. R⁴may be hydrogen or R¹⁵-substituted or unsubstituted alkyl, and R³ may behydrogen or R²³-substituted or unsubstituted alkyl. R³ and R⁴ mayoptionally be joined together with X to form a 4-7 membered (e.g. 5-7)membered heterocycloalkyl (e.g. an R²³ substituted species thereof).

In some embodiments, R⁷ is R⁸-substituted or unsubstituted heteroaryl,or -L⁶-R^(7A). L⁴ may be —C(O)—, and R^(7A) is R⁸-substituted orunsubstituted heteroaryl. R⁷, R⁸, L⁶, R^(7A) are as defined above.

In some embodiments, in the compound having the structure of Formula Ior II, R¹ is R⁷-substituted phenyl. In some related embodiments, R⁷ isR⁸-substituted or unsubstituted heteroaryl or -L⁶-R^(7A). L⁶ may be—C(O)—, and R^(7A) may be R⁸-substituted or unsubstituted heteroaryl. Infurther embodiments of Formula II, R³ and R⁴ are hydrogen.

In some embodiments, R⁷ is R⁸-substituted or unsubstituted purinyl,R⁸-substituted or unsubstituted pyrimidinyl, R⁸-substituted orunsubstituted imidazolyl, R⁸-substituted or unsubstituted1H-pyrrolo[2,3-b]pyridinyl, R⁸-substituted or unsubstituted pyrimidinyl,R⁸-substituted or unsubstituted 1H-indazolyl, or R⁸-substituted orunsubstituted 7H-pyrrolo[2,3-d]pyrimidinyl. R⁷ may also beR⁸-substituted or unsubstituted pyrrolopyrimidinyl, R⁸-substituted orunsubstituted indolyl, R⁸-substituted or unsubstituted pyrazolyl,R⁸-substituted or unsubstituted indazolyl, R⁸-substituted orunsubstituted imidazolyl, R⁸-substituted or unsubstituted thiazolyl,R⁸-substituted or unsubstituted benzothiazolyl, R⁸-substituted orunsubstituted oxazolyl, R⁸-substituted or unsubstituted benzimidazolyl,R⁸-substituted or unsubstituted benzoxazolyl, R⁸-substituted orunsubstituted isoxazolyl, R⁸-substituted or unsubstitutedbenzisoxazolyl, R⁸-substituted or unsubstituted triazolyl,R⁸-substituted or unsubstituted benzotriazolyl, R⁸-substituted orunsubstituted quinolinyl, R⁸-substituted or unsubstituted isoquinolinyl,R⁸-substituted or unsubstituted quinazolinyl, R⁸-substituted orunsubstituted pyrimidinyl, R⁸-substituted or unsubstituted pyridinylN-oxide, R⁸-substituted or unsubstituted furanyl, R⁸-substituted orunsubstituted thiophenyl, R⁸-substituted or unsubstituted benzofuranyl,R⁸-substituted or unsubstituted benzothiophenyl, R⁸-substituted orunsubstituted imidazo[1,2b]pyridazinyl. In some embodiments, R¹ isR⁸-substituted or unsubstituted 6,5 fused ring heteroaryl,R⁸-substituted or unsubstituted 5,6 fused ring heteroaryl,R⁸-substituted or unsubstituted 5,5 fused ring heteroaryl, orR⁸-substituted or unsubstituted 6,6 fused ring heteroaryl. In otherembodiments, R¹ is a R⁸-substituted or unsubstituted 5 or 6 memberedheteroaryl having at least 2 (e.g. 2 to 4) ring nitrogens.

In certain embodiments, R⁷ is -L⁴-R^(7A) and R^(7A) is R⁸-substituted orunsubstituted 1H-pyrrolo[2,3-b]pyridinyl.

In certain embodiments, X is N, and R¹ is R⁷-substituted 6-memberedheterocycloalkyl. R⁷ may be R⁸-substituted or unsubstituted heteroaryl.R¹ may be R⁷-substituted piperidinyl. R⁷ may be R⁸-substituted orunsubstituted purinyl, R⁸-substituted or unsubstituted pyrimidinyl,R⁸-substituted or unsubstituted imidazolyl, R⁸-substituted orunsubstituted 1H-pyrrolo[2,3-b]pyridinyl, R⁸-substituted orunsubstituted pyrimidinyl, R⁸-substituted or unsubstituted 1H-indazolyl,or R⁸-substituted or unsubstituted 7H-pyrrolo[2,3-d]pyrimidinyl. Incertain embodiments, R³ and R⁴ are hydrogen.

In yet further embodiments, R¹ is R⁷-substituted or unsubstituted 6,5fused ring heteroaryl, or R⁷-substituted or unsubstituted 5,6 fused ringheteroaryl. R⁷ may be —NH₂, R⁸-substituted or unsubstituted alkyl,R⁸-substituted or unsubstituted aryl, and R⁸ is independently —OH orunsubstituted alkyl. In other embodiments, R¹ is R⁷-substituted orunsubstituted indazolyl, or R⁷-substituted or unsubstitutedR¹-pyrrolo[2,3-d]pyrimidinyl. In certain embodiments, R³ and R⁴ arehydrogen.

In certain embodiments of compounds having the structure of Formula I orII, R¹ is R⁷-substituted or unsubstituted indazole, or R⁷-substituted orunsubstituted 7H-pyrrolo[2,3-d]pyrimidinyl, R³ is unsubstituted alkyl,and R⁴ is hydrogen. In further embodiments, R³ is phenylmethyl, and R⁴is hydrogen. In some embodiments, R³ and R⁴ join with N to form aR²³-substituted or unsubstituted pyrrolidinyl.

In certain embodiments contemplating compounds having structure ofFormula I or II, X is O, R¹ is R⁷-substituted or unsubstituted 6,5 fusedring heteroaryl, or R⁷-substituted or unsubstituted 5,6 fused ringheteroaryl. R⁷ may be —NH₂, R⁸-substituted or unsubstituted alkyl, orR⁸-substituted or unsubstituted aryl. R⁸ may be —OH or unsubstitutedalkyl, and R³ may be unsubstituted alkyl. In certain furtherembodiments, R¹ is R⁷-substituted 7H-pyrrolo[2,3-d]pyrimidine, and R⁷ is—NH₂, R⁸-substituted or unsubstituted alkyl, or R⁸-substituted orunsubstituted phenyl.

In some embodiments, the —C(O)X(L⁴-R³)_(z)(L⁵-R⁴) substituent of FormulaII is an electron withdrawing group. Therefore, the—C(O)X(L⁴-R³)_(z)(L⁵-R⁴) is capable of withdrawing negative charge fromthe olefin moiety to which it is attached. In some embodiments, the—C(O)X(L⁴-R³)_(z)(L⁵-R⁴) is capable of sufficiently withdrawing negativecharge from the olefin moiety to which it is attached to allow a thioladduct to form between an olefin carbon and the sulfhydryl of a kinaseactive site cysteine as discussed herein.

In certain embodiments of Formula I and Formula II, R¹ is substituted orunsubstituted heteroaryl. In other embodiments, R¹ is substituted orunsubstituted pyrazolopyrimidinyl (e.g. R⁷-substituted or unsubstitutedpyrazolopyrimidinyl). In other embodiments, R¹ is substituted orunsubstituted pyrrolopyrimidinyl (e.g. R⁷-substituted or unsubstitutedpyrrolopyrimidinyl).

In some embodiments, the kinase inhibitor has the structure:

In Formula IIIa, z is an integer from 1 to 4. L¹, L² and R⁷ are asdefined above. In some embodiments, L¹ is a bond.

In some embodiments, the kinase inhibitor has the structure:

In Formula IIIb, z is an integer from 1 to 4. L¹, L², R³, R⁴ and R⁷ areas defined above. In some embodiments, L¹ is a bond.

In some embodiments, the kinase inhibitor has the structure:

In Formula IIIc, W is —C(O)— or —S(O)₂—, z is an integer from 1 to 4.L¹, R³, R⁴ and R⁷ are as defined above. In some embodiments, L¹ is abond.

In some embodiments, the kinase inhibitor has the structure:

In Formula IIId, W is —C(O)— or —S(O)₂—, z is an integer from 1 to 4.L′, L², R³, R⁴ and R⁷ are as defined above. In some embodiments, L′ is abond. Ins some embodiments. In some embodiment, the kinase inhibitor iscompound 43, 44, 45, 46, 47, 48, 49 and 50.

In some embodiments, the kinase inhibitor has the structure:

In Formula IIIe, W is —C(O)— or —S(O)₂—, w is an integer from 1 to 5(e.g. 1). L¹, L², R³, R⁴, R⁷ and R⁸ are as defined above.

In another embodiment of Formula I or Formula II, L′ is substituted orunsubstituted arylene. In some embodiments, L′ is substituted orunsubstituted phenylene.

In some embodiments, the kinase inhibitor has the structure:

In Formula IVa, R¹, L², E and R¹¹ are as defined above. The symbol q isan integer from 1 to 4. In some embodiments R¹¹ is hydrogen. In someembodiments, R¹ is substituted or unsubstituted heteroaryl. In furtherembodiments, R¹ is substituted or unsubstituted pyrrolopyrimidine. Incertain embodiments of Formula IVa, R¹ is substituted or unsubstitutedheteroaryl, q is 0, and L¹ is substituted or unsubstituted phenylene.

In some embodiments, the kinase inhibitor has the structure:

In Formula IVb, R⁷, L², E and R¹¹ are as defined above. The symbol q isan integer from 0 to 4. In some embodiments R¹¹ is hydrogen. The symbolz is an integer from 1 to 4.

In some embodiments, the kinase inhibitor has the structure:

In Formula IVc, R⁷, L², and E are as defined above. The symbol z is aninteger from 1 to 4. In further embodiments, E is —NR³R⁴ (as definedabove).

In some embodiments, the kinase inhibitor has the structure:

In Formula IVd, R⁷, L², R³ and R⁴ are as defined above. The symbol z isan integer from 1 to 4. In further embodiments, L² is —C(O)—.

In some embodiments, the kinase inhibitor has the structure:

In Formula IVe, W is —C(O)— or —S(O)₂—, R⁷, R³ and R⁴ are as definedabove. The symbol z is an integer from 1 to 4. In some embodiments, R⁷is independently —NH₂, or substituted or unsubstituted aryl. In someembodiments, one of R⁷ is —NH₂, and another R⁷ is substituted aryl.

In some embodiments, the kinase inhibitor has the structure:

In Formula IVf, W is —C(O)— or —S(O)₂—, R⁷, R³ and R⁴ are as definedabove. In certain further embodiments, R³ and R⁴ are independentlyhydrogen, unsubstituted or substituted alkyl, or joined together to forma substituted or unsubstituted heteroalkyl. R⁷ may be substituted orunsubstituted phenyl. In some embodiments, the kinase inhibitor iscompound 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61.

In some embodiments, the kinase inhibitor has the structure:

In Formula (Va), R¹, L² and E are as defined above. In certainembodiments, E is —NR³R⁴ as defined above). L² may be —C(O)—.

In some embodiments, the kinase inhibitor has the structure:

In Formula Vb, W is —C(O)— or —S(O)₂—, R¹, R³ and R⁴ are as definedabove. In certain embodiments, R¹ is substituted alkyl.

In some embodiments, the kinase inhibitor has the structure:

In Formula Vc, W is —C(O)— or —S(O)₂—, R³ and R⁴ are as defined above.In certain embodiments, the kinase inhibitor is compound 38 or 39.

In some embodiments, R¹ is substituted or unsubstituted indazolyl, andthe kinase inhibitor has the structure of Formula VIa following, whereinR⁷ is as defined herein.

In some embodiments, the kinase inhibitor has the formula:

In Formula VIa, L², E and R⁷ are as defined above. The symbol z is aninteger from 1 to 4.

In some embodiments, the kinase inhibitor has the formula:

In Formula VIb, W is —C(O)— or —S(O)₂—, R³, R⁴ and R⁷ are as definedabove. The symbol z is an integer from 1 to 4.

In some embodiments, the kinase inhibitor has the formula:

In Formula VIc, W is —C(O)— or —S(O)₂—, R³, R⁴ and R⁷ are as definedabove. In some embodiments, the kinase inhibitor is compound 37, 40, 41or 42.

In another embodiment of Formula I, L¹ is substituted or unsubstitutedheterocycloalkylene, L² is —C(O)—. In further embodiments, R¹ issubstituted or unsubstituted heteroaryl, E is —NR³R⁴. In someembodiments, L¹ is piperidinyl. In further embodiments, R¹ is purinyl.

In some embodiments, the kinase inhibitor has the formula:

In Formula (VII), W is —C(O)— or —S(O)₂—, R³ and R⁴ are as definedabove. In some embodiments, the kinase inhibitor is compound 36.

The kinase inhibitor may be a reversible kinase inhibitor (as discussedherein). In some embodiments, the kinase inhibitor is a reversibledenatured kinase inhibitor (as discussed herein). In some embodiments,the kinase inhibitor is a covalent reversible kinase inhibitor (asdiscussed herein). In other embodiments, the kinase inhibitor is acovalent reversible denatured kinase inhibitor (as discussed herein).And in certain embodiments, the kinase inhibitor is a thiol covalentreversible denatured kinase inhibitor (as discussed herein).

In some embodiments, the compounds of the Formulae provided herein, andembodiments thereof, are stable at pH 7.5 (e.g. in phosphate-bufferedsaline at 37° C.). In some embodiments, where the compound of Formula Ior II, and embodiments thereof, are stable at pH 7.5, the compound has ahalf life of greater than 6 hours, 12 hours, 24 hours, or 48 hours. Insome embodiments, where the compounds of the Formulae provided herein,and embodiments thereof, are stable at pH 7.5, the compound has a halflife of greater than 12 hours. In some embodiments, where the compoundsof the Formulae provided herein, and embodiments thereof, are stable atpH 7.5, the compound has a half life of greater than 24 hours. In someembodiments, where the compounds of the Formulae provided herein, andembodiments thereof, are stable at pH 7.5, the compound has a half lifeof greater than 48 hours. In certain embodiments, the compounds of theFormulae provided herein, and embodiments thereof, exhibit kinaseinhibition within a cell. In some embodiments, the cell is a prokaryoteor eukaryote. The cell may be a eukaryote (e.g. protozoan cell, fungalcell, plant cell or an animal cell). In some embodiments, the cell is amammalian cell such as a human cell, cow cell, pig cell, horse cell, dogcell and cat cell, mouse cell, or rat cell. In some embodiments, thecell is a human cell. The cell may form part of an organ or an organism.In certain embodiments, the cell does not form part of an organ or anorganism.

In some embodiments, each substituted group described above in thecompounds of the Formulae provided herein is substituted with at leastone substituent group. More specifically, in some embodiments, eachsubstituted alkyl, substituted heteroalkyl, substituted cycloalkyl,substituted heterocycloalkyl, substituted aryl, substituted heteroaryl,substituted alkylene, substituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene described above in thecompounds of the Formulae provided herein is substituted with at leastone substituent group. In other embodiments, at least one or all ofthese groups are substituted with at least one size-limited substituentgroup. Alternatively, at least one or all of these groups aresubstituted with at least one lower substituent group.

In other embodiments of the compounds of the Formulae provided herein,each substituted or unsubstituted alkyl is a substituted orunsubstituted C₁-C₂₀ alkyl, each substituted or unsubstitutedheteroalkyl is a substituted or unsubstituted 2 to 20 memberedheteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₄-C₈ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 4 to 8membered heterocycloalkyl, each substituted or unsubstituted alkylene isa substituted or unsubstituted C₁-C₂₀ alkylene, each substituted orunsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20membered heteroalkylene, each substituted or unsubstituted cycloalkylenesubstituted or unsubstituted C₄-C₈ cycloalkylene, and each substitutedor unsubstituted heterocycloalkylene is a substituted or unsubstituted 4to 8 membered heterocycloalkylene.

Alternatively, each substituted or unsubstituted alkyl is a substitutedor unsubstituted C₁-C₈ alkyl, each substituted or unsubstitutedheteroalkyl is a substituted or unsubstituted 2 to 8 memberedheteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl, each substituted or unsubstituted alkylene isa substituted or unsubstituted C₁-C₈ alkylene, each substituted orunsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8membered heteroalkylene, each substituted or unsubstituted cycloalkylenesubstituted or unsubstituted C₅-C₆ cycloalkylene, and each substitutedor unsubstituted heterocycloalkylene is a substituted or unsubstituted 5to 7 membered heterocycloalkylene.

In some embodiments, the compounds of the Formulae provided herein isone or more of the compounds set forth in Table 1 and or Tables 2a-2ebelow. In other embodiments, the compound is one or more of thefollowing:

In some embodiments, the kinase inhibitor has the structure of FormulaVIII:

In Formula VIII and other Formulae provided herein, ring A is asubstituted or unsubstituted heteroaryl, such as an R³¹-substituted orunsubstituted heteroaryl. R¹ and L¹ are as defined above.

R³¹ is R^(23A) as defined above, or is hydrogen, halogen, —CN, —OH,—NH₂, —COOH, —CF₃, R³³-substituted or unsubstituted alkyl,R³³-substituted or unsubstituted heteroalkyl, R³³-substituted orunsubstituted cycloalkyl, R³³-substituted or unsubstitutedheterocycloalkyl, R³³-substituted or unsubstituted aryl, orR³³-substituted or unsubstituted heteroaryl.

R³³ is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R³⁴-substituted or unsubstituted alkyl, R³⁴-substituted or unsubstitutedheteroalkyl, R³⁴-substituted or unsubstituted cycloalkyl,R³⁴-substituted or unsubstituted heterocycloalkyl, R³⁴-substituted orunsubstituted aryl, or R³⁴-substituted or unsubstituted heteroaryl.

R³⁴ is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R³⁵-substituted or unsubstituted alkyl, R³⁵-substituted or unsubstitutedheteroalkyl, R³⁵-substituted or unsubstituted cycloalkyl,R³⁵-substituted or unsubstituted heterocycloalkyl, R³⁵-substituted orunsubstituted aryl, or R³⁵-substituted or unsubstituted heteroaryl.

R³⁵ is independently halogen, —CN, —OH, —NH₂, —COOH, —CF₃, unsubstitutedalkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstitutedheteroaryl.

In some embodiments, the kinase inhibitor has the structure of FormulaIX:

In Formula IX, ring A is a substituted or unsubstituted heteroaryl, suchas an R³¹-substituted or unsubstituted heteroaryl as set fort above. R¹and L¹ are as defined above.

In some embodiments of Formulae VIII and IX above, ring A isR³¹-substituted or unsubstituted heteroaryl. In some embodiments ofFormulae VIII and IX above, ring A is five-membered R³¹-substituted orunsubstituted heteroaryl or six-membered R³¹-substituted orunsubstituted heteroaryl.

In some embodiments, the kinase inhibitor has the structure of FormulaXa:

In Formula Xa, X₁ is —C(R³¹R³²)—, —N(R³¹)—, —S— or —O— when X₁ is notthe point of attachment. When X₁ is the point of attachment, then X₁ isC(R³¹) or N. X₂, X₃, X₄ and X₅ are independently —C(R³¹)═ or —N═ whennot the point of attachment. When X₂, X₃, X₄ or X₅ is the point ofattachment, then the X₂, X₃, X₄ or X₅ that is the point of attachment isC. At least one of X₁, X₂, X₃, X₄ and X₅ is not carbon (i.e. not—C(R³¹R³²)—, C(R³¹) or —C(R³¹)═ as appropriate). For example, in someembodiments at least one of X₁, X₂, X₃, X₄ and X₅ is nitrogen (i.e.—N(R³¹)— or —N═ as appropriate). Typically, at least 2 of X₁, X₂, X₃, X₄and X₅ is a carbon (e.g. —C(R³¹R³²)—, C(R³¹) or —C(R³¹)═).

R³¹ is as defined above. R³² is hydrogen, halogen, —CN, —OH, —NH₂,—COOH, —CF₃, R³³-substituted or unsubstituted alkyl, R³³-substituted orunsubstituted heteroalkyl, R³³-substituted or unsubstituted cycloalkyl,R³³-substituted or unsubstituted heterocycloalkyl, R³³-substituted orunsubstituted aryl, or R³³-substituted or unsubstituted heteroaryl. R³³is defined as disclosed above.

In some embodiments, the kinase inhibitor has the structure of FormulaXb:

In Formula Xb', X₁ is C(R³¹) or N. X₂, X₃, X₄ and X₅ are independently—C(R³¹)═ or —N═, provided, however, that at least one of X₁, X₂, X₃, X₄and X₅ is N. Typically, at least 2 of X₁, X₂, X₃, X₄ and X₅ is a carbon.In Formula Xb″, X₂ is C. X₁ is —C(R³¹R³²)—, —N(R³¹)—, —S— or —O—. X₃, X₄and X₅ are independently —C(R³¹)═ or —N═, provided, however, that atleast one of X₁, X₃, X₄ and X₅ is not carbon. L¹, R¹ and R³¹ are definedas disclosed above. Typically, at least one of X₁, X₃, X₄ and X₅ iscarbon.

In some embodiments, the kinase inhibitor has the structure of FormulaXc:

In Formula Xc, X₃ and X₅ are independently —C(R³¹)═. L¹, R¹ and R³¹ areas defined above.

In some embodiments, the kinase inhibitor has the structure of FormulaXd:

In Formula Xd, X₃, X₄ and X₅ are independently —C(R³¹)═. L¹, R¹ and R³¹are defined as disclosed above.

In some embodiments, the kinase inhibitor has the structure of FormulaXe:

In Formula Xe, X₄ and X₅ are independently —C(R³¹)═. X² is C. L¹, R¹ andR³¹ are as defined as disclosed above.

In some embodiments, the kinase inhibitor has the structure of FormulaXf:

In Formula Xf, X₄ and X₅ are independently —C(R³¹)═. L¹, R¹ and R³¹ aredefined as disclosed above.

In some embodiments, the kinase inhibitor has the structure of FormulaXIa:

In Formula XIa, X₆, X₇, X₈, X₉ and X₁₀ are independently —C(R³¹)═, —N═,or +N—O—, provided, however, that at least one of X₆, X₇, X₈, X₉ and X₁₀is N or +N—O—. L¹, R¹ and R³¹ are defined as disclosed above.

In some embodiments, the kinase inhibitor has the structure of FormulaXIb:

In Formula XIb, X₆, X₇, X₉ and X₁₀ are independently —C(R³¹)═. X₈ is Nor +N—O—. L¹, R¹ and R³¹ are defined as disclosed above.

In some embodiments, the kinase inhibitor has the structure of FormulaXIc:

In Formula XIc, X₆, X₈, X₉ and X₁₀ are independently —C(R³¹)═. X₇ is Nor +N—O—. L¹, R¹ and R³¹ are defined as disclosed above.

In some embodiments of Formulae VIII and IX above, ring A isR³¹-substituted or unsubstituted furyl, R³¹-substituted or unsubstitutedthienyl, R³¹-substituted or unsubstituted pyrrolyl, R³¹-substituted orunsubstituted imidazolyl, R³¹-substituted or unsubstituted pyrazolyl,R³¹-substituted or unsubstituted oxazolyl, R³¹-substituted orunsubstituted isoxazolyl, R³¹-substituted or unsubstituted thiazolyl,R³¹-substituted or unsubstituted isothiazolyl, R³¹-substituted orunsubstituted triazolyl, R³¹-substituted or unsubstituted oxadiazolyl,R³¹-substituted or unsubstituted pyridyl, R³¹-substituted orunsubstituted pyrimidyl, R³¹-substituted or unsubstituted pyridazinyl,R³¹-substituted or unsubstituted pyrrolinyl, R³¹-substituted orunsubstituted pyrazinyl, R³¹-substituted or unsubstituted tetrazolyl,R³¹-substituted or unsubstituted furanyl, R³¹-substituted orunsubstituted dihydrothieno-pyrazolyl, R³¹-substituted or unsubstitutedthianaphthenyl, R³¹-substituted or unsubstituted carbazolyl,R³¹-substituted or unsubstituted benzothienyl, R³¹-substituted orunsubstituted benzofuranyl, R³¹-substituted or unsubstituted indolyl,R³¹-substituted or unsubstituted quinolinyl, R³¹-substituted orunsubstituted benzotriazolyl, R³¹-substituted or unsubstitutedbenzothiazolyl, R³¹-substituted or unsubstituted benzooxazolyl,R³¹-substituted or unsubstituted benzimidazolyl, R³¹-substituted orunsubstituted isoquinolinyl, R³¹-substituted or unsubstitutedisoindolyl, R³¹-substituted or unsubstituted acridinyl, R³¹-substitutedor unsubstituted benzoisazolyl. R³¹ is defined as disclosed above.

In some embodiments of Formulae VIII, IX, Xa-Xf, and XIa-XIc above, R¹is R⁷-substituted or unsubstituted heterocycloalkyl, R⁷-substituted orunsubstituted aryl, or R⁷-substituted or unsubstituted heteroaryl. L¹ isa bond.

In some embodiments, a compound of Formulae I is one or more compoundsset forth in Table 1, Tables 2a-2e below, or the following compounds:

Using chemical synthesis techniques generally known in the art and thesynthesis techniques set forth in the Examples section, a person havingordinary skill in the art would be able to synthesize the compounds ofFormula I. In another aspect, a method of making a reversible kinaseinhibitor is provided. The method includes the step of modifying a knownnon-reversible or irreversible kinase inhibitor to include a substituenthaving the formula:

W, L², L⁴, L⁵, E, R³, R⁴, ring A, X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉,X₁₀, and z are as defined above. The symbol

represents the point of attachment of the substituent to the remained ofthe compound or solid support.

In another aspect, there is provided a protein adduct comprising aprotein bound to a kinase inhibitor provided herein. In someembodiments, the adduct has the formula:

The symbols in the protein adduct formulae (e.g. W, E, R¹, R³, R⁴, R⁷,R¹¹, ring A, X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, etc.) are asdefined above. The symbol Q represents a protein (e.g. peptide). Thesulfur attached to the Q typically forms part of a cysteine amino acid.In some embodiments, the cysteine linked to the inhibitor compounds isCys-481 of BTK, Cys-909 of JAK3, or Cys-436 of RSK2.

III. Methods of Inhibiting Protein Kinases

In another aspect, methods of inhibiting protein kinases are provided.The methods include contacting a protein kinase with an effective amountof a kinase inhibitor provided herein. The kinase inhibitor may have thestructure of the Formulae provided herein (or any of the embodimentsthereof described above). In some embodiments, the methods of inhibitinga protein kinase are conducted within a cell. Thus, in certainembodiments, methods of inhibiting a protein kinase within a cell areprovided. The method includes contacting a cell with an effective amountof a kinase inhibitor provided herein. The kinase inhibitor may have thestructure of the Formulae provided herein (or any of the embodimentsthereof described above). In some embodiments, the cell is a prokaryoteor eukaryote. The cell may be a eukaryote (e.g. protozoan cell, fungalcell, plant cell or an animal cell). In some embodiments, the cell is amammalian cell such as a human cell, cow cell, pig cell, horse cell, dogcell and cat cell, mouse cell, or rat cell. In some embodiments, thecell is a human cell. The cell may form part of an organ or an organism.In certain embodiments, the cell does not form part of an organ or anorganism.

The kinase inhibitor may be a reversible kinase inhibitor. A reversiblekinase inhibitor is a kinase inhibitor, as disclosed herein (e.g. thecompounds of the Formulae provided herein and embodiments thereof), iscapable of measurably dissociating from the protein kinase when theprotein kinase is intact (i.e. not denatured) or denatured (e.g.partially denatured or fully denatured). A “denatured” kinase is akinase without sufficient tertiary or secondary structure sufficient toretain kinase activity. An “intact” kinase is a kinase with sufficienttertiary or secondary structure sufficient to retain kinase activity.Therefore, in some embodiments, the method of inhibiting a proteinkinase includes contacting a protein kinase with a reversible kinaseinhibitor and allowing the reversible kinase inhibitor to reversiblybind to an active site cysteine residue, thereby inhibiting the proteinkinase.

In some embodiments, the reversible kinase inhibitor measurablydissociates from the protein kinase only when the protein kinase isdenatured, but does not measurably dissociate from the protein kinasewhen the protein kinase is intact (or dissociates at least 1, 1×10²,1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰ fold slowerrelative to the dissociation when the protein kinase is denatured). Areversible kinase inhibitor that measurably dissociates (or dissociatesat least 1, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10, 1×10⁸, 1×10⁹,1×10¹⁰ fold slower relative to the dissociation when the protein kinaseis denatured) from the protein kinase only when the protein kinase isdenatured, but does not measurably dissociate from the protein kinasewhen the protein kinase is intact is referred to herein as a “reversibledenatured kinase inhibitor.” After dissociating from the kinase, thereversible denatured kinase inhibitor can bind to the same or anotherkinase.

In certain embodiments, the method of inhibiting the protein kinaseincludes contacting the protein kinase with a kinase inhibitor whereinthe kinase inhibitor inhibits the protein kinase with an inhibitionconstant of less than 100 nM. And where the protein kinase inhibitor isa reversible protein kinase inhibitor, the method of inhibiting theprotein kinase includes contacting the protein kinase with a reversiblekinase inhibitor wherein the reversible kinase inhibitor inhibits theprotein kinase with an inhibition constant of less than 100 nM.

Where a kinase (also referred to herein as a protein kinase) isinhibited using a kinase inhibitor described herein, it is meant thatkinase activity (i.e. phosphorylation of a substrate molecule (e.g. aprotein substrate)) is decreased when contacted with the kinaseinhibitor relative to the activity of the kinase in the absence of thekinase inhibitor. In some embodiments, the kinase inhibitor decreasesthe kinase activity 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 100, 250,500, 1000, 5000, 10,000, 100,000, 500,000, 1,000,000, or more fold. Insome embodiments, the kinase inhibitor inhibits the activity of thekinase with an inhibition constant (KO of less than 100 μM, 5 μM, 1 μM,500 nM, 250 nM, 100 nM, 75 nM, 50 nM, 25 nM, 10 nM, 1 nM, 500 pM, 250pM, 100 pM, 75 pM, 50 pM, 25 pM, 10 pM, or 1 pM. In some embodiments,the kinase inhibitor inhibits the activity of the kinase with an IC₅₀ ofless than 100 μM, 10 μM, 5 μM, 1 μM, 500 nM, 250 nM, 100 nM, 75 nM, 50nM, 25 nM, 10 nM, 1 nM, 500 pM, 250 pM, 100 pM, 75 pM, 50 pM, 25 pM, 10pM, or 1 pM, when measured under the conditions set forth in theexamples section.

Where a reversible kinase inhibitor provided herein reversibly binds toan active site cysteine residue, a reversible bond is formed between theactive site cysteine residue and the reversible kinase inhibitor. Thereversible bond is typically a covalent bond. A “covalent reversiblekinase inhibitor,” as used herein, refers to a reversible kinaseinhibitor that forms a covalent bond with the kinase. Where the covalentreversible kinase inhibitor forms a reversible bond with an active sitecysteine residue, the covalent reversible kinase inhibitor is referredto herein as a “thiol covalent reversible kinase inhibitor.” In someembodiments, the covalent reversible kinase inhibitor measurablydissociates from the protein kinase only when the protein kinase isdenatured, but does not measurably dissociate (or dissociates at least1, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰ foldslower relative to the dissociation when the protein kinas is fully orpartially denatured) from the protein kinase when the protein kinase isintact (referred to herein as a “covalent reversible denatured kinaseinhibitor”). In some embodiments, the protein kinase is denatured (i.e.not intact) when placed in denaturing solution, such as 6 N guanidine,1% SDS, 50% MeCN, or similar protein denaturant, for second or minutes(e.g. 30 to 120 seconds, such as 60 seconds). A covalent reversibledenatured kinase inhibitor that forms a reversible bond with an activesite cysteine residue is termed a “thiol covalent reversible denaturedkinase inhibitor.”

In some embodiments, the thiol covalent reversible kinase inhibitorforms a bond between the cysteine sulfhydryl groups and a carbon atomforming part of the carbon-carbon double bond (i.e. olefin) of thecompound of the Formulae provided herein. Thus, in some embodiments,electrons of the sulfur atom of the active site cysteine sulfhydrylgroup attacks an electron deficient carbon atom of the carbon-carbondouble bond (olefin). In some embodiments, the electron deficient carbonatom of the carbon-carbon double bond is distal to the electronwithdrawing cyano group and the electron withdrawing -L²-E substituentof the kinase inhibitor of the Formulae provided herein and embodimentsthereof (i.e. the carbon attached to -L¹-R¹). In this way, a thioladduct is formed (e.g., Michael reaction with cysteine). Therefore, insome embodiments, the combination of cyano and -L²-E electronwithdrawing groups bound to the olefinic moiety increases the reactivityof the olefin to form a thiol adduct with the active site cysteineresidue. In some embodiments, the resulting thiol adduct is stable atabout pH 2 to about pH 7 (e.g. about pH 3). In some embodiments, thereversible kinase inhibitors described herein, after covalently bindingto the kinase active site cysteine residue as described herein, iscapable of dissociating from the kinase within seconds or minutes afterdenaturing/unfolding the kinase with 6 N guanidine, 1% SDS, 50% MeCN, orsimilar protein denaturant.

In some embodiments, in addition to increasing the reactivity of theolefin towards the active site cysteine sulfhydryl group, the cyano and-L²-E electron withdrawing groups function to increase the reversibilityof thiol adduct that is formed. Thus, in some embodiments, thereversible kinase inhibitors set forth herein is completely reversible.The term “completely reversible” means the reversible kinase inhibitorexhibits a measurable dissociation rate under conditions in which thekinase is not denatured. In some embodiments, the kinase inhibitorsprovided herein are not completely reversible (i.e. do not exhibit ameasurable dissociation rate under conditions in which the kinase isintact). Dissociation may be measured using any appropriate means,including dialysis and mass spectrometry. Specific methods of measuringdissociation are set forth in the Examples section below.

In some embodiments, the reversible denatured kinase inhibitor bindsreversibly to cellular components other than the protein kinase that thereversible denatured kinase inhibitor inhibits (or specificallyinhibits). The cellular components may be GSH, proteins or proteinfragments that are not targeted kinases (e.g. a kinase that does notinclude an active site cysteine or does not include an active sitecysteine within sufficient proximity to an ATP binding site), proteinfragments of targeted kinases (e.g. a kinase that has been digested suchthat the number of bonding points to the kinase reversible denaturedkinase inhibitor has been decreased such that the reversible denaturedkinase inhibitor dissociates from the kinase). Thus, in someembodiments, the reversible denatured kinase inhibitor measurablydissociates from the kinase where the kinase is partly or fullydigested. The ability of a reversible denatured kinase inhibitor tomeasurably dissociate from cellular components other than the intact offull length protein kinase that the reversible denatured kinaseinhibitor inhibits may provide decreased toxicity, including decreasedimmunogenic toxicity. In certain embodiments, the -L¹-R¹ group of thereversible denatured kinase inhibitor is a kinase ATP binding sitemoiety and the electron deficient olefin carbon binds to a sulfhydryl ofa kinase active site cysteine. Thus, in some embodiments the kinaseinhibitors provided herein bind to at least two points of the proteinkinase: at least one residue within the ATP binding site moiety and asulfhydryl of a kinase active site cysteine.

In some embodiments, physiological concentrations of glutathione (e.g. 5or 10 mM GSH) have little or no measurable effect on the ability of thereversible kinase inhibitors (e.g. provided herein to inhibit a proteinkinase (e.g. eversible denatured kinase inhibitor only reversibly bindsto GSH thereby enabling increased binding to the target kinase). In someembodiments, the IC50 or K_(i) of the reversible kinase inhibitor isincreased no more than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, 0.5%, 0.1%, 0.01% or 0.001% in the presence of physiologicalconcentrations of glutathione(e.g. 5 or 10 mM GSH). In otherembodiments, the IC50 or K_(i) of the reversible kinase inhibitor is notmeasurably increased by the presence of physiological concentrations ofglutathione (e.g. 5 or 10 mM GSH). In some embodiments, thephysiological concentrations of glutathione (e.g. 5 or 10 mM GSH) havelittle or no measurable effect on the ability of the reversible kinaseinhibitors provided herein to inhibit a protein kinase wherein thereversible kinase inhibitor is present at low concentrations (e.g. lessthan 100 nM, 75 nM, 50 nM, 25 nM, 10 nM, 5 nM, 3 nM, 1 nM, 500 pM, 250pM, 100 pM, 75 pM, 50 pM, 25 pM, 10 pM, or 1 pM). In some embodiments,the physiological concentrations of glutathione (e.g. 5 or 10 mM GSH)have little or no measurable effect on the ability of the reversiblekinase inhibitors provided herein to inhibit a protein kinase whereinthe reversible kinase inhibitor is present at a concentration of lessthan 10 nM, 5 nM, 4 nM 3 nM, 2 nM or 1 nM. In certain embodiments, thephysiological concentrations of glutathione (e.g. 5 or 10 mM GSH) havelittle or no measurable effect on the ability of the reversible kinaseinhibitors provided herein to inhibit a protein

In some embodiments, physiological concentrations of adenosinetriphosphate (e.g. 1 mM ATP) have little or no measurable effect on theability of the reversible kinase inhibitors provided herein to inhibit aprotein kinase. In some embodiments, the IC50 or K_(i) of the reversiblekinase inhibitor is increased no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%,3%, 2%, 1%, 0.5%, 0.1%, 0.01% or 0.001% in the presence of physiologicalconcentrations of adenosine triphosphate (e.g. 1 mM ATP). In otherembodiments, the IC50 or K₁ of the reversible kinase inhibitor is notmeasurably increased by the presence of physiological concentrations ofadenosine triphosphate (e.g. 1 mM ATP).

In certain embodiments, the reversible kinase inhibitors provided hereinreacts reversibly with GSH. In certain embodiments, the reversiblekinase inhibitors provided herein react rapidly and reversibly with GSH.Thus, in certain embodiments, the reversible kinase inhibitors providedherein react reversibly with GSH (e.g. rapidly and reversibly) whilealso reversibly binding to an active site cysteine residue (e.g. at aconcentration of less than 10 nM, 5 nM, 4 nM 3 nM, 2 nM or 1 nM). TheGSH may be at physiological concentration (e.g. 5-10 mM). Without beingbound by any particular mechanistic theory, it is believed that theability of reversible kinase inhibitors provided herein to react rapidlyand reversibly with cellular glutathione protects most non-targetedcellular proteins from the electrophilic qualities of the reversiblekinase inhibitor.

The protein kinase may be any appropriate kinase. In some embodiments,the protein kinase includes a cysteine residue in the active site. Aprotein kinase active site is a portion of the protein kinase in whichthe protein kinase substrate is phosphorylated. The kinase active siteis typically a pocket or cleft containing amino acid residues that bindto a substrate (also referred to herein as kinase active site bindingresidues) and amino acid residues that participate in the catalyticphosphorylation reaction (also referred to herein as kinase active sitecatalytic residues). The reversible kinase inhibitors provided hereinare capable of inhibiting the kinase catalytic action by fitting intothe kinase active site and disrupting the ability of the kinase tophosphorylate the substrate. The active sites of many protein kinasesare known in the art through structure determinations (e.g. X-raycrystallography or three dimensional NMR techniques). Where the threedimensional structure has not been determined, the structure of anactive site of a protein kinase may be determined by the primary aminoacid sequence using computer software modeling programs generally knownin the art.

Protein kinases inhibited using the kinase inhibitors provided hereininclude, but are not limited to, serine/threonine-specific proteinkinases, tyrosine-specific protein kinases, receptor tyrosine kinases,receptor-associated tyrosine kinases, histidine-specific proteinkinases, and aspartic acid/glutamic acid-specific protein kinases, asknown in the art. In some embodiments, the kinase is a tyrosine proteinkinase or serine/threonine protein kinases. Examples of kinases includeSRC, YES, FGR, CHK2, FGFR1—4, BTK, EGFR, HER2, HER4, HER3, JAK3, PLK1-3,MPSI, RON, MEK1/2, ERK1/2, VEGFR, KIT, KDR, PDGFR, FLT3, CDK8, MEK7,ROR1, RSK1-4, MSK1/2, MEKK1, NEK2, MEK5, MNK1/2, MEK4, TGFbR2, ZAP70,WNK1-4, BMX, TEC, TXK, ITK, BLK, MK2/3, LIMK1, TNK1, CDK11, p70S6 Kb,EphB3, ZAK, and NOK.

IV. Methods of Treating Disease

In another aspect, a method of treating a disease associated with kinaseactivity in a subject in need of such treatment. The method includesadministering to the subject an effective amount (e.g. a therapeuticallyeffective amount) of a compound having the structure of the Formulaeprovided herein (or an embodiment thereof as described above).

In some embodiments, the disease associated with kinase activity ischronic disease. The disease may be cancer, epilepsy, HIV infection,autoimmune disease (e.g. arthritis), ischemic disease (e.g. heart attackor stroke), stroke, neurodegenerative diseases, metabolic orinflammation. In certain embodiments, the disease is cancer , including,for example, leukemia, carcinomas and sarcomas, such as cancer of thebrain, breast, cervix, colon, pancreas, head & neck, liver, kidney,lung, non-small cell lung, prostate, melanoma, mesothelioma, ovary,sarcoma, stomach, uterus and medulloblastoma. Additional examplesinclude, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma,neuroblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis,primary macroglobulinemia, primary brain tumors, malignant pancreaticinsulanoma, malignant carcinoid, urinary bladder cancer, premalignantskin lesions, testicular cancer, lymphomas, thyroid cancer,neuroblastoma, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms ofthe endocrine and exocrine pancreas. In some embodiments, the disease isliver cancer, colon cancer, breast cancer, melanoma, acute myelogenousleukemia, chronic myelogenous leukemia, or nonsmall-cell lung cancer. Insome embodiments, the disease is cancers which have metastasized. Insome embodiments, the disease is rheumatoid arthritis, systemic lupuserythematosus, multiple sclerosis, scleroderma or polymyositis. In someembodiments, the disease is diabetes, obesity, or lipid disorders. Insome embodiments, the disease may be caused by an infectious agent suchas caused by bacteria, parasite or virus. In some embodiments, thedisease is acute such as myocardial infarction, stroke or asthma. Insome embodiments the disease is Parkinson's disease or amyotrophiclateral sclerosis.

V. Assays

Using techniques known in the art and the guidance provided herein,candidate kinase inhibitors may be easily assayed for their ability toinhibit any known protein kinase. For example, candidate kinaseinhibitors having the structure of the Formulae provided herein orembodiments thereof may be first assayed using computer modelingtechniques in order to assess potential binding contacts between kinaseactive site binding residues and/or kinase active site catalyticresidues. Such computer modeling techniques may also be referred to asin silico techniques. As discussed above, the kinase active site bindingresidues and/or kinase active site catalytic residues are known oreasily determined for any kinase in which the primary amino acidstructure is known. In particular, computer modeling techniques may beemployed to assess the ability of candidate kinase inhibitors to reactwith a kinase active site cysteine residue with the electron deficientolefin carbon to form a thiol adduct. For example, where the kinaseinhibitor electron deficient olefin carbon is within 10 Å of the kinaseactive site cysteine sulfhydryl, the potency and/or selectivity ofkinase inhibitor may be improved (e.g. by 1000-10,000-fold).

Likewise, computer modeling techniques may be used to assess the abilityof candidate kinase inhibitors to fit into the kinase active sitewithout creating stearic clashes. As described above, in someembodiments, —R¹ or -L-R¹ to fit within the kinase ATP binding siteand/or make contacts with amino acid residues within the kinase ATPbinding site. Therefore, computer modeling techniques may be used toassess the ability of —R¹ or -L-R¹ to fit within the kinase ATP bindingsite and/or make contacts with amino acid residues within the kinase ATPbinding site. The computer modeling assays described above may be usedto assess the kinase inhibition ability of candidate kinase inhibitorshaving different general chemical scaffolds within the structure of theFormulae provided herein or embodiments thereof. In this way, newclasses of chemical scaffolds may be assessed using computer modelingprior to performing in vitro activity assays.

In vitro assays may also be used to assess the kinase inhibitingproperties of candidate kinase inhibitors having the structure of theFormulae provided herein or embodiments thereof. In vitro kinase assaysare well known in the art. High throughput techniques are known anduseful for quickly assessing large numbers of kinase inhibitorcandidates using binding assays for a large number of kinase panels.See, for example, Karaman et al., Nat. Biotechnol. 2008 January;26(1):127-32.

Compounds that decrease kinase catalytic activity may also be identifiedand tested using biologically active protein kinases, either recombinantor naturally occurring. Protein kinases can be found in native cells,isolated in vitro, or co-expressed or expressed in a cell. Certainprotein kinases specifically phosphorylate particular substrates. Wherespecific substrates are known, the ability of a candidate kinaseinhibitor to reduce phosphorylation of the specific substrate may beassayed. General, or non-specific, kinase substrates may also beemployed.

The kinase inhibitors provided herein may also be tested in vitro fortheir ability to inhibit a mutant of a kinase that does not contain anactive site cysteine. The ability of a kinase inhibitor to decrease thecatalytic activity of a kinase having an active site cysteine while nothaving the ability (or having measurably decreased ability) to decreasethe catalytic activity of a mutant of the kinase that does not containan active site cysteine is indicative of a kinase inhibitor thatinhibits the kinase by binding to the active cite cysteine. For example,the C436V mutant of RSK2 may be resistant to certain kinase inhibitors(IC50>10 uM) that show strong inhibitory activity against the wild typeRSK2. This result supports the conclusion that RSK2 inhibition requiresthe formation of a covalent bond between Cys436 and the inhibitor.

As described above, E and -L²-E are typically substituents thatsufficiently withdraw electrons from the reaction center olefin carbonto reversibly bind to the sulfhydryl of a kinase active cite cysteine(e.g. when the kinase is partly or fully denatured). The kinaseinhibitors provided herein may also be tested in vitro for their abilityto reversibly bind to the active site cysteine of a protein kinase bymeasuring association and dissociation of the kinase inhibitor from theprotein kinase (e.g. partially or fully denatures) or from a thiolcompound (e.g. 2-mercaptoethanol (BME)). The ability of the reactioncenter carbon of a kinase inhibitor provided herein to reversibly bindto the sulfhydryl of a kinase active cite cysteine may be measured usingany appropriate means, including dialysis, mass spectrometry, NMR and UVdetection (see Examples section for more details). For example, thekinase inhibitors may be assayed by detecting the binding of a thiolcompound such as BME. The binding may be assessed using UV detection ofcompounds that typically become less UV active upon binding to a thiolcompound or by detecting the binding using proton NMR. Typically, theassays are conducted by titering in the thiol compound and examining achange in the endpoint binding detection parameter (e.g. UV activity orproton NMR). Reversibility is assessed by dilution. Specific examplesare provided below in the Examples section (see Example 82).

The kinase inhibitors provided herein may also be tested in vitro fortheir stability at pH 7.5. Any appropriate method may be used todetermine the stability of a kinase inhibitor set forth herein at pH7.5. Appropriate methods include, for example, LC-MS (e.g. HPLC-MS) aswell as measuring changes in UV absorption where the kinase inhibitorincludes a chromophore group. UV absorption may be measured usinghigh-throughput techniques (e.g. multiwell plated for scanning largenumbers of kinase inhibitors simultaneously). Stability may be assessedusing phosphate-buffered saline at pH 7.5 at 37° C. Compounds havinghalf-lives greater than 6 hours, 12 hours, 24 hours, or 48 hours may bemay be selected.

Cellular assays may also be used to assess the kinase inhibitingproperties of candidate kinase inhibitors having the structure of theFormulae provided herein or embodiments thereof. Cellular assays includecells from any appropriate source, including plant and animal cells(such as mammalian cells). The cellular assays may also be conducted inhuman cells. Cellular assays of kinase inhibition are well known in theart, and include methods in which a kinase inhibitor is delivered intothe cell (e.g. by electroporation, passive diffusion, microinjection andthe like) and a kinase activity endpoint is measured, such as the amountof phosphorylation of a cellular substrate, the amount of expression ofa cellular protein, or some other change in the cellular phenotype knownto be affected by the catalytic activity of the particular kinase beingmeasured. For example, phosphorylation of a particular cellularsubstrate may be assessed using a detection antibody specific or thephosphorylated cellular substrate followed by western blottingtechniques and visualization using any appropriate means (e.g.fluorescent detection of a fluorescently labeled antibody).

Measuring the reduction in the protein kinase catalytic activity in thepresence of a kinase inhibitor disclosed herein relative to the activityin the absence of the inhibitor may be performed using a variety ofmethods known in the art, such as the assays described in the Examplessection below. Other methods for assaying the activity of kinaseactivity are known in the art. The selection of appropriate assaymethods is well within the capabilities of those having ordinary skillin the art.

Once kinase inhibitors are identified that are capable of reducingkinase catalytic activity in vitro and/or in a cell, the compounds maybe further tested for their ability to selectively inhibit kinaseactivity in animal models (e.g. whole animals or animal organs). Thus,kinase inhibitors may be further tested in cell models or animal modelsfor their ability to cause detectable changes in phenotype related to aparticular kinase activity. In addition to cell cultures, animal modelsmay be used to test inhibitors of kinases for their ability to treat,for example, cancer in an animal model.

VI. Pharmaceutical Formulations

In another aspect, the present invention provides pharmaceuticalcompositions comprising a kinase inhibitor compound of the invention ora kinase inhibitor compound in combination with a pharmaceuticallyacceptable excipient (e.g. carrier).

The pharmaceutical compositions include optical isomers, diastereomers,or pharmaceutically acceptable salts of the inhibitors disclosed herein.For example, in some embodiments, the pharmaceutical compositionsinclude a compound of the present invention and citrate as apharmaceutically acceptable salt. The kinase inhibitor included in thepharmaceutical composition may be covalently attached to a carriermoiety, as described above. Alternatively, the kinase inhibitor includedin the pharmaceutical composition is not covalently linked to a carriermoiety.

A “pharmaceutically suitable carrier,” as used herein refers topharmaceutical excipients, for example, pharmaceutically,physiologically, acceptable organic, or inorganic carrier substancessuitable for enteral or parenteral application which do notdeleteriously react with the extract. Suitable pharmaceuticallyacceptable carriers include water, salt solutions (such as Ringer'ssolution), alcohols, oils, gelatins and carbohydrates such as lactose,amylose or starch, fatty acid esters, hydroxymethycellulose, andpolyvinyl pyrrolidine. Such preparations can be sterilized and, ifdesired, mixed with auxiliary agents such as lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, and/or aromatic substances and the likewhich do not deleteriously react with the compounds of the invention.

The compounds of the invention can be administered alone or can becoadministered to the patient. Coadministration is meant to includesimultaneous or sequential administration of the compounds individuallyor in combination (more than one compound). Thus, the preparations canalso be combined, when desired, with other active substances (e.g. toreduce metabolic degradation).

A. Formulations

The kinase inhibitors of the present invention can be prepared andadministered in a wide variety of oral, parenteral and topical dosageforms. Thus, the compounds of the present invention can be administeredby injection (e.g. intravenously, intramuscularly, intracutaneously,subcutaneously, intraduodenally, or intraperitoneally). Also, thecompounds described herein can be administered by inhalation, forexample, intranasally. Additionally, the compounds of the presentinvention can be administered transdermally. It is also envisioned thatmultiple routes of administration (e.g., intramuscular, oral,transdermal) can be used to administer the compounds of the invention.Accordingly, the present invention also provides pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier orexcipient and one or more compounds of the invention.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers can be eithersolid or liquid. Solid form preparations include powders, tablets,pills, capsules, cachets, suppositories, and dispersible granules. Asolid carrier can be one or more substance, which may also act asdiluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material.

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

The powders and tablets preferably contain from 5% to 70% of the activecompound. Suitable carriers are magnesium carbonate, magnesium stearate,talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoabutter, and the like. The term “preparation” is intended to include theformulation of the active compound with encapsulating material as acarrier providing a capsule in which the active component with orwithout other carriers, is surrounded by a carrier, which is thus inassociation with it. Similarly, cachets and lozenges are included.Tablets, powders, capsules, pills, cachets, and lozenges can be used assolid dosage forms suitable for oral administration.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

When parenteral application is needed or desired, particularly suitableadmixtures for the compounds of the invention are injectable, sterilesolutions, preferably oily or aqueous solutions, as well as suspensions,emulsions, or implants, including suppositories. In particular, carriersfor parenteral administration include aqueous solutions of dextrose,saline, pure water, ethanol, glycerol, propylene glycol, peanut oil,sesame oil, polyoxyethylene-block polymers, and the like. Ampules areconvenient unit dosages. The compounds of the invention can also beincorporated into liposomes or administered via transdermal pumps orpatches. Pharmaceutical admixtures suitable for use in the presentinvention include those described, for example, in PharmaceuticalSciences (17th Ed., Mack Pub. Co., Easton, Pa.) and WO 96/05309, theteachings of both of which are hereby incorporated by reference.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents.

Also included are solid form preparations, which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

The quantity of active component in a unit dose preparation may bevaried or adjusted from 0.1 mg to 10000 mg, more typically 1.0 mg to1000 mg, most typically 10 mg to 500 mg, according to the particularapplication and the potency of the active component. The compositioncan, if desired, also contain other compatible therapeutic agents.

Some compounds may have limited solubility in water and therefore mayrequire a surfactant or other appropriate co-solvent in the composition.Such co-solvents include: Polysorbate 20, 60 and 80; Pluronic F-68, F-84and P-103; cyclodextrin; and polyoxyl 35 castor oil. Such co-solventsare typically employed at a level between about 0.01% and about 2% byweight.

Viscosity greater than that of simple aqueous solutions may be desirableto decrease variability in dispensing the formulations, to decreasephysical separation of components of a suspension or emulsion offormulation and/or otherwise to improve the formulation. Such viscositybuilding agents include, for example, polyvinyl alcohol, polyvinylpyrrolidone, methyl cellulose, hydroxy propyl methylcellulose,hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propylcellulose, chondroitin sulfate and salts thereof, hyaluronic acid andsalts thereof, combinations of the foregoing. Such agents are typicallyemployed at a level between about 0.01% and about 2% by weight.

The compositions of the present invention may additionally includecomponents to provide sustained release and/or comfort. Such componentsinclude high molecular weight, anionic mucomimetic polymers, gellingpolysaccharides and finely-divided drug carrier substrates. Thesecomponents are discussed in greater detail in U.S. Pat. Nos. 4,911,920;5,403,841; 5,212,162; and 4,861,760. The entire contents of thesepatents are incorporated herein by reference in their entirety for allpurposes.

B. Effective Dosages

Pharmaceutical compositions provided by the present invention includecompositions wherein the active ingredient is contained in atherapeutically effective amount, i.e., in an amount effective toachieve its intended purpose. The actual amount effective for aparticular application will depend, inter alia, on the condition beingtreated. For example, when administered in methods to treat cancer, suchcompositions will contain an amount of active ingredient effective toachieve the desired result (e.g. decreasing the number of cancer cellsin a subject).

The dosage and frequency (single or multiple doses) of administered to amammal can vary depending upon a variety of factors, including a diseasethat results in increased activity of kinase, whether the mammal suffersfrom another disease, and the route of administration; size, age, sex,health, body weight, body mass index, and diet of the recipient; natureand extent of symptoms of the disease being treated (e.g., cancer), typeof concurrent treatment, complications from the disease being treated orother health-related problems. Other therapeutic regimens or agents canbe used in conjunction with the methods and compounds of the invention.

For any compound described herein, the therapeutically effective amountcan be initially determined from cell culture assays. Targetconcentrations will be those concentrations of active compound(s) thatare capable of reducing the level of kinase catalytic activity, asmeasured, for example, using the methods described.

Therapeutically effective amounts for use in humans may be determinedfrom animal models. For example, a dose for humans can be formulated toachieve a concentration that has been found to be effective in animals.The dosage in humans can be adjusted by monitoring kinase inhibition andadjusting the dosage upwards or downwards, as described above.

Dosages may be varied depending upon the requirements of the patient andthe compound being employed. The dose administered to a patient, in thecontext of the present invention, should be sufficient to affect abeneficial therapeutic response in the patient over time. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side-effects. Generally, treatment is initiated with smallerdosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under circumstances is reached. In one embodiment of theinvention, the dosage range is 0.001% to 10% w/v. In another embodiment,the dosage range is 0.1% to 5% w/v.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compound effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned which does not causesubstantial toxicity and yet is entirely effective to treat the clinicalsymptoms demonstrated by the particular patient. This planning shouldinvolve the careful choice of active compound by considering factorssuch as compound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration and the toxicity profile of the selected agent.

C. Toxicity

The ratio between toxicity and therapeutic effect for a particularcompound is its therapeutic index and can be expressed as the ratiobetween LD₅₀ (the amount of compound lethal in 50% of the population)and ED₅₀ (the amount of compound effective in 50% of the population).Compounds that exhibit high therapeutic indices are preferred.Therapeutic index data obtained from cell culture assays and/or animalstudies can be used in formulating a range of dosages for use in humans.The dosage of such compounds preferably lies within a range of plasmaconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. See, e.g. Fingl etal., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch.1, p. 1, 1975.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition and theparticular method in which the compound is used.

D. Additional Agents and Therapeutic Modalities

In some embodiments, the kinase inhibitors provided herein may be usedin combination with other therapeutic agents or therapeutic modalities.In some embodiments, the additional therapeutic agent is an anticanceragent. The therapeutic agent may be a chemotherapeutic agents, abiologic agent, hormonal therapy agent, or a kinase inhibitor that isnot a kinase inhibitor of the Formulae provided herein (or embodimentsthereof). The additional therapeutic agent may additionally be analkylating agent, an anthracylcines, a monoclonal antibody, a cytokine,a nucleoside analog, prednisone, a taxane, estrogen, progesterone,hormone antagonists, a vinca alkaloid, an anti-metabolites or the like.

In some embodiments, the kinase inhibitors provided herein may be usedin combination with other therapeutic modalities such as radiationtherapy and surgery.

VII. Examples

The following examples are offered to illustrate, but not to limit theclaimed invention.

General Chemistry Methods.

Low-resolution electrospray ionization mass spectra (ESI⁺-MS) wererecorded on a Waters Micromass ZQ 4000 spectrometer. LC/MS (MS: ESI⁺)was performed on a Waters AllianceHT LC/MS with a flow rate of 0.2 mlmin⁻¹ (monitored at 210 nm, 260 nm, and/or 350 nm) using an Xterra MSC18 column (Waters). For air- and water-sensitive reactions, glasswarewas oven- or flame-dried prior to use and reactions were performed underargon. Dichloromethane, dimethylformamide, methanol, tetrahydrofuran,toluene, and diisopropylamine were dried using the solvent purificationsystem manufactured by Glass Contour, Inc. (Laguna Beach, Calif.). Allother solvents were of ACS chemical grade (Fisher) and used withoutfurther purification unless otherwise indicated. Analytical andpreparative thin layer chromatography were performed with silica gel 60F₂₅₄ glass plates (EM Science). Flash chromatography was conducted with230-400 mesh silica gel (Selecto Scientific). Preparative highperformance liquid chromatography (HPLC) was performed on a Prostar 210(Varian) with a flow rate of 10 ml min⁻¹ (monitored at 210 nm and 260nm) using a COMBI-A C18 preparatory column (Peeke Scientific).

General Procedure for JAK2 and JAK3 Kinase Assays.

Active JAK2 (Human, residues 808-end) and JAK3 (Human, residues 781-end)was purchased from Millipore. Active kinase (3 nM) in 8 mM HEPES, pH7.0, 200 uM EDTA, 10 mM MgCl₂, 0.2 mg/mL BSA, 100 uM ATP and 10 mM GSHwere pre-incubated with inhibitors (eight or ten concentrations, induplicate) for 30 minutes at room temperature. Kinase reactions wereinitiated by the addition of 0.3 μCi/μL of γ-³²P-ATP (6000 Ci/mmol, NEN)and 100 uM peptide substrate (JAK3-tide for JAK3 or PDK-tide for JAK2,Millipore) and incubated for 30 minutes at room temperature. Kinaseactivity was determined by spotting 5 μL of each reaction onto sheets ofphosphocellulose. Each blot was washed once with 1% AcOH solution, twicewith 0.1% H₃PO₄ solution, and once with MeOH (5-10 minutes per wash).Dried blots were exposed for 30 minutes to a storage phosphor screen andscanned by a Typhoon imager (GE Life Sciences). The data were quantifiedusing the SPOT program (Knight, Z. et al. Nature Protocols, 2 (10),2459-66) and plotted using GraphPad Prism 4.0 software.

General Procedure for Btk Kinase Assay.

Btk (human, full length) was purchased (Invitrogen, catalog numberPV3363) and used as specified in the product literature. Btk (2 nM finalconcentration) was pre-incubated with inhibitors (six or tenconcentrations, in duplicate) for 30 minutes at room temperature. Kinasereactions were initiated by the addition of 0.16 μCi/μL of γ-³²P-ATP(6000 Ci/mmol, NEN) and 0.2 mg/mL substrate (poly[Glu:Tyr], 4:1 Glu:Tyr)and incubated for 30 minutes at room temperature. Kinase activity wasdetermined by spotting 6 μL of each reaction onto sheets ofphosphocellulose. Each blot was washed once with 1% AcOH solution, twicewith 0.1% H₃PO₄ solution, and once with MeOH (5-10 minutes per wash).Dried blots were exposed for 30 minutes to a storage phosphor screen andscanned by a Typhoon imager (GE Life Sciences). The data were quantifiedusing ImageQuant (v. 5.2, Molecular Dynamics) and plotted using GraphPadPrism 4.0 software.

Example 1 3-(4-(9H-purin-6-yl)phenyl)-2-cyanoacrylamide

To a solution of 1-tetrahydropyran-1-yl-6-(p-formylphenyl)purine(Donald, A. et al. J. Med. Chem. 2007, 50, 2289-2292) (170 mg, 0.55mmol) in 6 mL THF was added 20 drops of aqueous 1 N HCl. The reactionwas stirred at room temperature overnight. After 18 hours a whiteprecipitate had formed, and all starting material had been consumed asdetermined by TLC. The reaction was diluted with EtOAc, washed withsaturated NaHCO₃, and the organic layer was dried with Na₂SO₄. Rotaryevaporation afforded 100 mg (81%) of 2 as a light yellow solid that wascarried on without further purification. Exact mass: 224.07, M/z found:225.3 (M+H)⁺.

A slurry of the deprotected 6-(p-formylphenyl)-purine (10.9 mg, 0.049mmol), 2-cyanoacetamide (5.4 mg, 0.064 mmol), and PPh₃ (13 mg, 0.049mmol) in THF (0.5 mL) was heated to 80° C. in a sealed vial. The mixturewas stirred at 80° C. for 12 hours and concentrated in vacuo. Theresidue was chromatographed on silica with 9:1 CH₂Cl₂:MeOH, affording9.6 mg (87%) of 3-(4-(9H-purin-6-yl)phenyl)-2-cyanoacrylamide (mixtureof E/Z isomers) as a light yellow solid. Exact mass: 290.09, M/z found:291.3 (M+H)⁺.

Example 2 4-(1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzaldehyde

A slurry of 3-bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (100 mg, 0.29mmol), 4-formylphenyl boronic acid (66 mg, 0.44 mmol) and K₂CO₃ (126 mg,0.87 mmol) in 5:1 dioxane:water (2 mL) was degassed argon for 30 minutesin a microwave vial. Pd(PPh₃)₄ (33 mg, 0.029 mmol) was added and thevessel was purged with argon. The vial was heated to 150° C. for 15minutes in a microwave reactor, cooled to room temperature, diluted withEtOAc, washed with water, the organic layer dried with Na₂SO₄ andconcentrated in vacuo. The residue was chromatographed on silica with1:1 hexanes:EtOAc, affording 92 mg (86%) of4-(1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzaldehyde as a yellow solid.

Example 3 4-(1H-pyrrolo[2,3-b]pyridin-3-yl)benzaldehyde

To a solution of 4-(1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzaldehyde(162 mg, 0.45 mmol) in 2:1 THF:MeOH (2 mL) was added Cs₂CO₃ (435 mg,1.34 mmol). The solution turned yellow upon addition of the carbonatebase, and was stirred at room temperature for 18 hours. After completeconsumption of starting material, monitored by TLC, the light orangesolution was concentrated in vacuo, the residue taken up in 10 mL ofwater, and the resulting slurry stirred vigorously for 30 minutes. Theyellow precipitate was filtered, washed with water, and dried undervacuum to afford 74 mg of 4-(1H-pyrrolo[2,3-b]pyridin-3-yl)benzaldehydeas a bright yellow solid. Exact Mass: 222.08, M/z found: 223.3 (M+H)⁺.

Example 4 3-(4-(1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)-2-cyanoacrylamide

To a slurry of 4-(1H-pyrrolo[2,3-b]pyridin-3-yl)benzaldehyde (8 mg,0.036 mmol) in 1:1 THF:MeOH was added 2-cyanoacetamide (4 mg, 0.043mmol) and PPh₃ (5 mg, 0.018 mmol). The reaction mixture was heated to80° C. for 48 hours, and after significant evolution of product,monitored by LCMS, the reaction was concentrated and taken up in a smallvolume of THF. EtOH was added and the resulting yellow precipitatefiltered and dried in vacuo to afford 1.2 mg of3-(4-(1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)-2-cyanoacrylamide (mixtureof E/Z isomers) as a brilliant yellow solid. Exact mass: 288.30, M/zfound: 289.3 (M+H)⁺.

Example 5 3-(1H-pyrrolo[2,3-b]pyridine-3-carbonyl)benzaldehyde

7-azaindole (182 mg, 1.5 mmol) and AlCl₃ (945 mg, 7.4 mmol) werecombined in dry CH₂Cl₂ (50 mL) in a flame-dried round bottom flask underargon. The heterogeneous yellow mixture was allowed to stir at roomtemperature for one hour, at which time 3-formyl benzoyl chloride (505mg, 2.9 mmol) was added dropwise via syringe under positive pressure ofargon. The mixture slowly became a translucent yellow solution. Aftertwo additional hours of stirring at room temperature, MeOH (˜20 mL) wasadded slowly to quench excess acid chloride. The solvent was removed invacuo and the residue chromatographed on silica with 9:1 EtOAc:MeOH toafford 42 mg (11%) of3-(1H-pyrrolo[2,3-b]pyridine-3-carbonyl)benzaldehyde as a white solid.Exact mass: 250.07, M/z found: 251.3 (M+H)⁺.

Example 6 3-(3-(1H-pyrrolo[2,3-b]pyridine-3-carbonyl)phenyl)-2-cyanoacrylamide

To a solution of 3-(1H-pyrrolo[2,3-b]pyridine-3-carbonyl)benzaldehyde(19.5 mg, 0.08 mmol) in THF (1 mL) was added 2-cyanoacetamide (15 mg,0.17 mmol) and piperidine (10 μL, 0.096 mmol) and the slurry was stirredat 80° C. for three hours with evolution of a white precipitate. Theprecipitate was collected by filtration to afford 1.5 mg (6%) of3-(3-(1H-pyrrolo[2,3-b]pyridine-3-carbonyl)phenyl)-2-cyanoacrylamide(mixture of E/Z isomers) as a white solid. Exact mass: 316.10, M/zfound: 317.3 (M+H)⁺.

Example 7 2-cyano-3-(1H-indazol-5-yl)acrylamide

To a solution of 5-formyl-1H-indazole (102.5 mg, 0.7 mmol) in THF (2 mL)was added 2-cyanoacetamide (75 mg, 0.9 mmol) and piperidine (20 μL, 0.2mmol). The reaction was stirred for 18 hours at room temperature,followed by addition of a spatula-tip of cyanoacetamide. The reactionwas stirred for another three hours and yellow solids began toprecipitate. The reaction was filtered and the solids were washed withTHF and dried in vacuo to afford 65 mg (44%) of2-cyano-3-(1H-indazol-5-yl)acrylamide (mixture of E/Z isomers) as ayellow solid. Exact mass: 212.07, M/z found: 213.4 (M+H)⁺.

Example 8 3-(1H-indazol-5-yl)-2-(pyrrolidine-1-carbonyl)acrylonitrile

To a solution of 6-formyl-1H-indazole (115 mg, 0.8 mmol) in THF (2 mL)was added 2-cyano-N-isopropylacetamide (100 mg, 0.7 mmol) and DBU (1304,1.05 mmol). The resulting brown solution was stirred at room temperaturefor 18 hours, monitored by TLC. An additional spatula tip of2-cyano-N-isopropylacetamide was added, and the reaction stirred foranother 18 hours with minimal evolution of additional product. Thereaction was concentrated, taken up in a small volume of EtOAc, andpurified by preparative TLC to afford 43 mg (20%) of3-(1H-indazol-6-yl)-2-(pyrrolidine-1-carbonyl)acrylonitrile (mixture ofE/Z isomers) as an amorphous tan solid. Exact mass: 266.12, M/z found:267.5 (M+H)⁺.

Example 9 2-cyano-3-(1H-indazol-6-yl)acrylamide

To a solution of 6-formyl-1H-indazole (102.5 mg, 0.7 mmol) in THF (2 mL)was added 2-cyanoacetamide (78.2 mg, 0.7 mmol) and piperidine (504, 0.5mmol). The reaction was stirred for 18 hours at room temperature withproduction of a white precipitate. The reaction was filtered and thesolids were washed with THF and dried in vacuo to afford 69 mg (46%) of2-cyano-3-(1H-indazol-6-yl)acrylamide (mixture of E/Z isomers) as awhite solid. Exact mass: 212.07, M/z found: 213.4 (M+H)⁺.

Example 10 2-cyano-3-(1H-indazol-6-yl)-N-methylacrylamide

To a solution of 6-formyl-1H-indazole (103.5 mg, 0.7 mmol) in THF (1 mL)was added 2-cyano-N-methylacetamide (70 mg, 0.7 mmol) and DBU (130 μL,1.05 mmol). Immediately upon DBU addition, the yellow slurry became aclear orange solution, followed by rapid evolution of an orangeprecipitate. After an hour of stirring at room temperature, the reactionwas concentrated, taken up in a small volume of 1:1 EtOAc:H₂O andsonicated vigorously to break up the residue. The resulting solids werefiltered, washed with EtOAc and H₂O and dried to afford 21 mg (13%) of2-cyano-3-(1H-indazol-6-yl)-N-methylacrylamide (mixture of E/Z isomers)as a tan solid. Exact mass: 226.09, M/z found: 227.4 (M+H)⁺.

Example 11 2-cyano-3-(1H-indazol-6-yl)-N-isopropylacrylamide

To a solution of 6-formyl-1H-indazole (101.5 mg, 0.7 mmol) in THF (1 mL)was added 2-cyano-N-isopropylacetamide (99.9 mg, 0.8 mmol) and DBU (130μL, 1.05 mmol). After 1 hour, complete consumption of starting materialwas observed, monitored by TLC. The reaction was diluted with EtOAc andwashed with H₂O and brine. Hexanes was added slowly to the remainingorganic layer until a light precipitate formed, which was collected byfiltration to afford 48 mg (27%) of2-cyano-3-(1H-indazol-6-yl)-N-isopropylacrylamide (mixture of E/Zisomers) as a white solid.

Example 12 (1-(9H-purin-6-yl)piperidin-4-yl)methanol

6-Chloropurine (509 mg, 3.2 mmol), 4-piperidine methanol (737 mg, 6.4mmol) and Et₃N (2.25 mL, 25.6 mmol) was dissolved in n-BuOH (30 mL) andheated to 100° C. in a sealed vessel. The light orange solution washeated overnight, cooled to room temperature, and concentrated to afforda tan solid. The residue was triturated with 3:1 hexanes:MeOH (10 mL)and dried in vacuo to afford 401 mg (57%) of(1-(9H-purin-6-yl)piperidin-4-yl)methanol as a bright white solid.

Example 13 1-(9H-purin-6-yl)piperidine-4-carbaldehyde

(1-(9H-purin-6-yl)piperidin-4-yl)methanol (250 mg, 1.1 mmol) andDess-Martin periodinane (700 mg, 1.65 mmol) were combined in dry CH₂Cl₂(30 mL) and the slurry stirred vigorously until it became a yellow,heterogeneous solution. Water (20 μL) in CH₂Cl₂ (20 mL) was addeddropwise to the reaction, which became cloudy upon addition of ˜1 molarequivalent of water. After an additional 30 minutes of vigorousstirring, the reaction mixture was diluted in EtOAc (˜100 mL) and washedwith 1:1 saturated NaS₂O₄:NaHCO₃, water, and brine. The aqueous layerwas back-extracted with EtOAc, the combined organic layers dried withNa₂SO₄ and concentrated. The residue was chromatographed on silica with9:1 CH₂Cl₂:MeOH to afford 124 mg (48%) of1-(9H-purin-6-yl)piperidine-4-carbaldehyde as a waxy, colorless solid.

Example 14 3-(1-(9H-purin-6-yl)piperidin-4-yl)-2-cyanoacrylamide

1-(9H-purin-6-yl)piperidine-4-carbaldehyde (11.2 mg, 0.048 mmol),2-cyanoacetamide (6 mg, 0.071 mmol) and PPh₃ (6 mg, 0.023 mmol) werecombined in THF (1 mL) and heated to 80° C. in a sealed vial. After 18hours of heating, a white precipitate had formed but TLC indicated thepresence of significant quantities of starting material. A spatula tipof 2-cyanoacetamide was added to the reaction and it was stirred for anadditional 18 hours at 80° C. The reaction was cooled and theprecipitate collected by filtration to afford 6 mg (46%) of3-(1-(9H-purin-6-yl)piperidin-4-yl)-2-cyanoacrylamide (mixture of E/Zisomers) as a white solid. Exact mass: 297.13, M/z found: 298.3 (M+H)⁺.

Example 15 2-(2-oxo-2-p-tolylethyl)isoindoline-1,3-dione

2-bromo-1-p-tolylethanone (20 g, 93.8 mmol) was dissolved in 100 mL DMF.Potassium phthalamide (19.9 g, 103.2 mmol) was added and the reactionwas stirred at room temperature for 3 hours. The reaction was diluted toa total volume of 200 mL with CH₃C1 and the product precipitated out ofsolution. The precipitated product was collected by filtration. Theleftover filtrate was concentrated, redissolved in 100 mL CH₃C1, andthen washed with water, saturated sodium bicarbonate and brine. Theaqueous layer was extracted with CH₂Cl₂+10% MeOH, and the resultingorganic layer was dried over Na₂SO₄ and concentrated in vacuo. The totalamount of 2-(2-oxo-2-p-tolylethyl)isoindoline-1,3-dione obtained was24.4 g (94% yield).

Example 16 2-amino-4-p-tolyl-1H-pyrrole-3-carbonitrile

To a solution of malononitrile (1.23 g, 18.6 mmol) in MeOH (22.5 ml)/48%w/v aq. NaOH (3.5 ml)/H₂O (7.5 ml) was added2-(2-oxo-2-p-tolylethyl)isoindoline-1,3-dione (4 g, 14.3 mmol). Thereaction mixture was stirred at room temperature for 3 hours after whichthe product precipitated out of solution. The solid was collected byfiltration and washed with water. To the leftover filtrate was addedwater and the resulting precipitation was filtered off and combined withthe product. The solid product was dried under vacuum overnight to give2.3 g (82% yield) of 2-amino-4-p-tolyl-1H-pyrrole-3-carbonitrile.

Example 17 methyl N-3-cyano-4-p-tolyl-1H-pyrrol-2-ylformimidate

To a solution of 2-amino-4-p-tolyl-1H-pyrrole-3-carbonitrile (2.3 g,11.6 mmol) in triethyl orthoformate (10 ml) was added acetic anhydride(110 μl, 1.1 mmol) and the mixture was refluxed for 1 hour. Aftercooling to room temperature, the solvent was removed in vacuo. The 2.88g (99% yield) of crude methylN-3-cyano-4-p-tolyl-1H-pyrrol-2-ylformimidate was azeotropically driedwith toluene and carried on directly to the next step.

Example 18 N′-(3-cyano-4-p-tolyl-1H-pyrrol-2-yl)formimidamide

The crude methyl N-3-cyano-4-p-tolyl-1H-pyrrol-2-ylformimidate (2.88 g,11.5 mmol) was dissolved in 7 N NH₃ in MeOH (80 ml) and stirred at roomtemperature in a capped round bottom flask for 4 hours. The solvent wasremoved in vacuo and the crude N′-(3-cyano-4-p-tolyl-1H-pyrrol-2-yl)formimidamide product was dried on high vacuum overnight to give 2.27 g(88% yield) of product.

Example 19 5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine

N′-(3-cyano-4-p-tolyl-1H-pyrrol-2-yl)formimidamide (2.27 g, 10.2 mmol)was dissolved in MeOH (30 ml) and heated to reflux. Sodium methoxide(1.5 m, 25% wt in MeOH, 5.1 mmol) was added and the reaction was stirredat reflux for 3 hours after which the product precipitated out ofsolution. After cooling to room temperature, the solid was isolated byfiltration and dried under high vacuum to give 2.3 g (100%) of pure5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine as a tan solid.

Example 207-(3-(tert-butyldimethylsilyloxy)propyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine

To a 0° C. slurry of 5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (5.01g, 22.3 mmol) in DMF (120 mL) was added NaH (0.98 g of 60% oildispersion, 24.6 mmol) and the resulting mixture was allowed to warm toroom temperature. After stirring for 30 min,3-(t-butyldimethylsilyloxy)propyl iodide (7.38 g, 24.6 mmol) was addeddropwise over 10 min. After stirring for an additional 4 hours thereaction was quenched by adding 100 ml water and the precipitatedproduct was filtered and dried under high vacuum to give 7.78 g (88%yield) of7-(3-(tert-butyldimethylsilyloxy)propyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine.Exact Mass 396.23. Found 397.5 [M+H]⁺.

Example 216-bromo-7-(3-(tert-butyldimethylsilyloxy)propyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine

To a solution of7-(3-(tert-butyldimethylsilyloxy)propyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine(1.22 g, 3 mmol) in DMF (20 ml) was added N-bromosuccinimide (0.65 g,3.6 mmol) and the mixture was stirred for 24 hours protected from light.The reaction was diluted with EtOAc (25 ml) and washed with water andbrine. The combined organic fractions were dried over anhydrous Na₂SO₄,filtered, concentrated and purified by flash column chromatography (40%ethyl acetate/hexanes) to give 0.9 g (62% yield) of6-bromo-7-(3-(tert-butyldimethylsilyloxy)propyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine. Exact Mass: 474.15. Found 475 [M]⁺ and 477[M+2]⁺.

Example 227-(3-(tert-butyldimethylsilyloxy)propyl)-5-p-tolyl-6-vinyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine

To a solution of6-bromo-7-(3-(tert-butyldimethylsilyloxy)propyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine(1.0 g, 2.1 mmol) in toluene (30 ml) was added tributylvinyltin (0.8 ml,2.73 mmol). Argon gas was bubbled through the solution for 10 min.Tetrakis(triphenylphosphine)palladium (244 mg, 0.21 mmol) was quicklyadded, and argon gas was bubbled through the solution for another 10min. The reaction was then stirred at reflux for 3 h. The reaction wasfiltered through Celite and the filtrate was concentrated. The productwas purified by flash column chromatography in 50% EtOAc/hexanes to givea light yellow residue that was lyophilized from benzene to give 757 mg(85% yield) of7-(3-(tert-butyldimethylsilyloxy)propyl)-5-p-tolyl-6-vinyl-7H-pyrrolo[2,3-d]pyrimidin-4-amineas a light yellow powder. Exact Mass 422.25. Found 423.1 [M+H]⁺.

Example 234-amino-7-(3-(tert-butyldimethylsilyloxy)propyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidine-6-carbaldehyde

To a solution of7-(3-(tert-butyldimethylsilyloxy)propyl)-5-p-tolyl-6-vinyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine(760 mg, 1.8 mmol) in 3:1 THF:H₂O (11.3 ml) under argon was dropwiseadded osmium tetroxide (1.75 ml, 0.18 mmol, 2.5% in t-BuOH). Thereaction was stirred at room temperature under argon for 20 min. Sodiumperiodate (860 mg, 3.6 mmol, dissolved in 2.4 ml warm water) was addeddropwise to the reaction over a period of 30 min. The reaction wasstirred for 1.5 h at room temperature and was then diluted with ethylacetate. The organic layer was washed with saturated aqueous sodiumthiosulfate and the aqueous layer was back extracted with ethyl acetate.The combined organic layers were dried over sodium sulfate, filtered andconcentrated. The residue was purified by flash column chromatography in50% EtOAc/hexanes to give 417 mg (55% yield) of a yellowish oil thatsolidified upon standing. Exact Mass 424.23. Found 425.1 [M+H]⁺.

Example 24 Methyl3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyanoacrylate

To a solution of4-amino-7-(3-(tert-butyldimethylsilyloxy)propyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidine-6-carbaldehyde(52 mg, 0.123 mmol) in dry THF (1.5 ml) was added DBU (22 μl, 0.147mmol) and methyl 2-cyanoacetate (13 mg, 0.135 mmol). The reaction wasstirred at room temperature for 1 hr after which conversion had reached90%. The reaction was concentrated and the residue was purified bypreparatory thin layer chromatography in 50% EtOAc/hexanes to give 51 mg(80% yield) of the TBS-protected cyanoacrylate as a yellow film. To asolution of the TBS-protected cyanoacrylate (51 mg, 0.098 mmol) in THF(1.5 ml) was added 1N aqueous HCl (500 μl). The reaction was stirred atroom temperature for 1 hr and then diluted with ethyl acetate. Theorganic layer was washed with saturated aqueous sodium bicarbonate andbrine, then dried over sodium sulfate, filtered, and concentrated. Theyellow residue was purified by precipitation from methanol/hexanes (1:3)to give 12 mg (25% yield) of methyl3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyanoacrylate(mixture of E/Z isomers) as a yellow solid. Exact Mass 391.16. Found392.0 [M+H]⁺.

Example 25 tert-butyl3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-c]pyrimidin-6-yl)-2-cyanoacrylate

To a solution of4-amino-7-(3-(tert-butyldimethylsilyloxy)propyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidine-6-carbaldehyde(100 mg, 0.235 mmol) in dry THF (2 ml) was added DBU (45 μA 0.306 mmol)and tert-butyl 2-cyanoacetate (34 μl, 0.235 mmol). The reaction wasstirred at room temperature for 2 hours after which it was diluted withethyl acetate. The organic layer was washed with aqueous 0.1 N HClsolution and brine, then dried over sodium sulfate, filtered, andconcentrated. The residue was purified by flash column chromatography in50% EtOAc/hexanes and then by HPLC purification (gradient 30-100% MeOHover 35 min, 10 ml min⁻¹ flow rate, retention time 9.34 min) to give21.3 mg (21% yield) of tert-butyl3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyanoacrylate(mixture of E/Z isomers). Chemical Formula: C₂₄H₂₇N₅O₃ Exact Mass433.21. Found 434.1 [M+H]⁴.

Example 263-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyanoacrylamide

To a solution of4-amino-7-(3-(tert-butyldimethylsilyloxy)propyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidine-6-carbaldehyde(120 mg, 0.283 mmol) in dry THF (2 ml) and isopropanol (1 ml) was addedpiperidine (28 μl, 0.283 mmol) and 2-cyanoacetamide (23 mg, 0.283 mmol).The reaction was stirred at room temperature for 16 hours after whichanother equivalent of 2-cyanoacetamide and piperidine were added. After10 days the reaction was diluted with ethyl acetate and the organiclayer was washed with water and then dried over sodium sulfate, filteredand concentrated. The crude residue was dissolved in 3 ml dry THF and1.2 ml of 1N HCl was added. The reaction was stirred for 1.5 hours, thendiluted with ethyl acetate. The organic layer was washed with saturatedaqueous sodium bicarbonate followed by brine and then dried over sodiumsulfate, filtered, and concentrated. The residue was purified by flashcolumn chromatography in 3-5% MeOH/DCM to give 38 mg (42% yield) of3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyanoacrylamide(mixture of E/Z isomers) as a yellow powder. Exact Mass: 376.16. Found377.4 [M+H]⁴.

Example 27 2-Cyano-N-benzylacetamide

To stirred solution of diisopropylethyl amine (506 μl, 3 mmol) andbenzylamine (327 μl, 3 mmol) in DMF was added cyanoacetic acid (85 mg, 1mmol), EDC (625 mg, 3 mmol), and HOBt (208 mg, 1.5 mmol). The reactionwas stirred room temperature overnight. The reaction was then dilutedwith ether and the organic layer was washed twice with 1N HCl and oncewith brine. The organic layer was dried over sodium sulfate, filteredand concentrated by rotary evaporation. The clear residue waslyophilized from benzene to give 24 mg (14% yield) of an off-whitepowder that needed no further purification.

Example 28 2-Cyano-N-isopropylacetamide

To stirred methyl cyanoacetate (2.0 g, 20 mmol) at 0° C. was addedisopropylamine (1.7 ml, 20 mmol) dropwise, keeping the temperature below10° C. The reaction was stirred at 0° C. for 1 h, after which 2 ml of15% aqueous NaOH were added and the reaction was stirred at roomtemperature for 5 min. The mixture was diluted with ethyl acetate andwashed with water. The organic layer was dried over sodium sulfate,filtered and concentrated to give 1.06 g (42% yield) of a white solidthat needed no further purification. Ref: Basheer, A. et. al. J. Org.Chem. (2007), 72: 5297-5312.

Example 293-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-N-benzyl-2-cyanoacrylamide

To a solution of4-amino-7-(3-(tert-butyldimethylsilyloxy)propyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidine-6-carbaldehyde(58 mg, 0.138 mmol) in dry THF (1 ml) was added piperidine (29 μA 0.276mmol) and N-benzyl cyanoacetamide (24 mg, 0.138 mmol). The reaction wasstirred at room temperature for 2 days after which conversion hadreached 50% and stopped. The reaction was concentrated and the residuewas purified by flash column chromatography in 50% EtOAc/hexanes to givea yellow powder that was dissolved in THF (1 ml) and 100 μl 1 N HCl wasadded. The reaction was stirred at room temperature for 1 hour, dilutedwith ethyl acetate, and the layers were separated. The organic layer waswashed with saturated aqueous sodium bicarbonate followed by brine andthen dried over sodium sulfate, filtered, and concentrated. The yellowoil was purified by flash column chromatography, eluting with 100% EtOActo 5% MeOH/EtOAc, to give a yellow residue that was lyophilized frombenzene to give 9.8 mg (46% yield) of3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-N-benzyl-2-cyanoacrylamide(mixture of E/Z isomers) as a yellow powder. Exact Mass: 466.21. Found:467.5 [M+H]⁺.

Example 303-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyano-N-isopropylayerylamide

To a solution of4-amino-7-(3-(tert-butyldimethylsilyloxy)propyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidine-6-carbaldehyde(100 mg, 0.236 mmol) in dry THF (1.5 ml) was added piperidine (50 μl,0.476 mmol) and N-isopropyl cyanoacetamide (60 mg, 0.476 mmol). Thereaction was stirred at room temperature for 4 days after whichconversion had reached 50%. The reaction was concentrated, and theresidue was purified by flash column chromatography in 50% EtOAc/hexanesto give a yellow foam that was dissolved in THF (1 ml) and 1 N HCl (100μl). The reaction was stirred at room temperature for 1.5 hours, dilutedwith ethyl acetate, and the layers were separated. The organic layer waswashed with saturated aqueous sodium bicarbonate followed by brine andthen dried over sodium sulfate, filtered and concentrated. The yellowoil was purified by flash column chromatography, eluting with 100% EtOActo 5% MeOH/EtOAc, to give a yellow oil that was lyophilized from benzeneto give 26 mg (54% yield) of3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyano-N-isopropylacrylamide(mixture of E/Z isomers) as a yellow powder. Exact Mass: 418.21. Found:419.5 [M+H]⁺.

Example 31 6-bromo-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine

5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (1.5 g, 6.7 mmol) wasdissolved in hot DMF and then allowed to cool to room temperature. Thecloudy suspension was stirred under argon and protected from light asNBS (1.4 g, 8.04 mmol) was added in portions over 20 minutes. One hourafter addition the solution went clear and a precipitate began to form.After 2 hours, the reaction was filtered and the solid was washed withDMF. The combined filtrates were concentrated and stirred in water for10 minutes to provide a second crop of solid. The grey solids werecombined to give 1.464 g (72% yield).

Example 32 5-p-tolyl-6-vinyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine

To a stirred solution of6-bromo-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (500 mg, 1.65 mmol)was added tributylvinylstannane (579 μl, 1.98 mmol) and the solution wasdegassed for 5 minutes. Palladium tetrakistriphenylphosphine (134 mg,0.116 mg) was added and the reaction was brought to reflux and stirredovernight. The reaction mixture was filtered through a pad of Celite,and the solid was washed 3 times with hot toluene. The filtrate wasconcentrated and the residue was recrystallized from EtOAc/hexanes togive 190 mg (46% yield) of a grey solid.

Example 33(E)-3-(4-amino-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyanoacrylamide

A 2.5% solution (w/v) of osmium tetroxide in tert-butanol (136 μl, 0.013mmol) was added dropwise to a stirred solution of5-p-tolyl-6-vinyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (190 mg, 0.68 mmol)in 3:1 THF/H₂O (5.2 ml). The suspension was stirred at room temperaturefor 10 minutes followed by the dropwise addition of NaIO₄ (262 mg, 1.224mmol) suspended in 1 ml of H₂O. The reaction was then stirred at roomtemperature overnight. The reaction was diluted with ethyl acetate andquenched with saturated aqueous sodium thiosulfate. The organic layerwas separated and the aqueous layer was extracted 3 times with ethylacetate. The combined organic layers were concentrated to give a yellowsolid that was dissolved in 2:1 isopropanol/THF (3 ml). To this solutionwas added cyanoacetamide (30 mg, 0.359 mmol) and piperidine (35 μl,0.359 mmol). The reaction was stirred at room temperature overnightduring which a yellow precipitate formed. This was filtered to give 46mg (41% yield, two steps) of2-cyano-3-(7-p-tolyl-5H-pyrrolo[3,2-d]pyrimidin-6-yl)acrylamide. ExactMass: 318.12. Found: 319.00 [M+H]⁺.

Example 34(E)-3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)acrylonitrile

To a flame-dried flask were added tert-butyl7-(3-(tert-butyldimethylsilyloxy)propyl)-6-formyl-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylcarbamate(218 mg, 0.416 mmol), (triphenylphosphoranylidene)acetonitrile (500.9mg, 1.664 mmol) and dry CH₂Cl₂ (10 ml). The reaction was stirred underargon at room temperature for 16 hours after which the reaction wasconcentrated and the crude residue was purified by flash columnchromatography in 25% EtOAc/hexanes to give 196 mg (87% yield) of3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)acrylonitrileas a mixture of isomers.3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)acrylonitrile(80 mg, 0.139 mmol) was dissolved in 2 ml CH₂Cl₂ and cooled to 0° C.after which 2 ml of TFA was added. The reaction was stirred at 0° C. for1 hour and then allowed to reach room temperature. After 16 hours, thereaction was concentrated and the crude residue was dissolved in 3 mldry THF and 1 ml of 1 N HCl was added. The reaction was stirred for 15hours, then diluted with ethyl acetate and the organic layer was washedwith saturated aqueous sodium bicarbonate, followed by brine, then driedover sodium sulfate, filtered, and concentrated. The residue waspurified by flash column chromatography in 3% MeOH/DCM to give 9.1 mg(19% yield) of(E)-3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)acrylonitrileand 24.6 mg (53% yield) of(Z)-3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)acrylonitrile.Chemical Formula C₁₉H₁₉N₅O Exact Mass: 333.16. Found 334.5 [M+H]⁺.

Example 353-(1-(9H-purin-6-yl)piperidin-4-yl)-2-cyano-N-methylacrylamide

1-(9H-purin-6-yl)piperidine-4-carbaldehyde (17 mg, 0.086 mmol),2-cyano-methylacetamide (11 mg, 0.112 mmol) and PPh₃ (28 mg, 0.106 mmol)were combined in THF (1 mL) and heated to 110 □C in a sealed vial. After18 hours of heating, the reaction mixture was diluted with EtOAc (2 mL)and washed with water (1 mL). The organic layer was concentrated andsubmitted to preparative TLC (eluting with 9:1 CH₂Cl₂:MeOH) to afford 6mg (23%) of3-(1-(9H-purin-6-yl)piperidin-4-yl)-2-cyano-N-methylacrylamide as awhite solid. Exact mass: 311.15, M/z found: 312.05 (M+H)+

Example 36 2-cyano-3-(1H-indazol-5-A-N,N-dimethylacrylamide

To a solution of 6-formyl-1H-indazole (101 mg, 0.7 mmol) in THF (1 mL)was added 2-cyano-N-dimethylacetamide (80 mg, 0.7 mmol) and DBU (67 μL,0.7 mmol). The resulting brown solution was stirred at room temperaturefor 18 hours, followed by 4 hours at 60° C. Starting material remained,so the reaction was stirred for another 18 hours with minimal evolutionof additional product. The reaction was concentrated, taken up in asmall volume of EtOAc and the resulting white precipitate was collectedby filtration to afford 30 mg of2-cyano-3-(1H-indazol-5-yl)-N,N-dimethylacrylamide (19%) as a whitesolid. Exact mass: 240.10, M/z found: 241.09 (M+H)⁺.

Example 373-(3-(1H-pyrrolo[2,3-b]pyridine-3-carbonyl)phenyl)-2-cyano-N-methylacrylamide

To a solution of 3-(1H-pyrrolo[2,3-b]pyridine-3-carbonyl)benzaldehyde(47 mg, 0.2 mmol) in THF (1 mL) was added 2-cyanoacetamide (19 mg, 0.2mmol) and DBU (20 μL, 0.2 mmol) and the slurry was stirred at roomtemperature for five minutes. A few drops of MeOH were added to thereaction to keep a gummy precipitate from sticking to the walls, and thereaction was stirred overnight with evolution of a yellowishprecipitate. The precipitate was collected by filtration to afford 8 mg(12%) of3-(3-(1H-pyrrolo[2,3-b]pyridine-3-carbonyl)phenyl)-2-cyano-N-methylacrylamideas a white solid. Exact mass: 330.11, M/z found: 331.05 (M+H)⁺.

Example 383-(3-(1H-pyrrolo[2,3-b]pyridine-3-carbonyl)phenyl)-2-cyano-N,N-dimethylacrylamide

To a solution of 3-(1H-pyrrolo[2,3-b]pyridine-3-carbonyl)benzaldehyde(47 mg, 0.2 mmol) in THF (1 mL) was added 2-cyanoacetamide (19 mg, 0.2mmol) and DBU (20 μL, 0.2 mmol) and the slurry was stirred at roomtemperature for several hours, at which point the brown slurry became aclear yellow solution. The following day, the reaction was concentratedto a brown, waxy solid, resuspended in EtOAc:MeOH (2:1), and theresultant precipitate was collected by filtration to afford 12 mg (17%)of3-(3-(1H-pyrrolo[2,3-b]pyridine-3-carbonyl)phenyl)-2-cyano-N,N-dimethylacrylamideas a white solid. Exact mass: 344.13, M/z found: 345.09 (M+H)⁺.

Example 39 3-(3,4,5-trimethoxyphenyl)-1H-indazole-5-carbaldehyde

3-iodo-5-formylindazole (210 mg, 0.735 mmol), trimethoxyphenyl boronicacid (187 mg, 0.882 mmol) and K₂CO₃ (309 mg, 2.21 mmol) were combined in5:1 Dioxane:H₂O (2 mL) and degassed with bubbling argon for 20 minutes.Pd(PPh₃)₄ (123 mg, 0.106 mmol) was added, and the reaction vessel purgedwith argon. The reaction was microwaved at 150° C. for fifteen minutes.The crude reaction mixture was diluted with EtOAc (20 mL), washed with1M HCl (10 mL), H₂O (10 mL) and brine (10 mL), dried with Na₂SO₄, andconcentrated. The residue was purified on silica, eluting with 3:2Hex:EtOAc, and the impure fractions containing product were concentratedto a yellow solid. This yellow solid was suspended in EtOAc and thewhite solids filtered to afford 140 mg (83%) of3-(3,4,5-trimethoxyphenyl)-1H-indazole-5-carbaldehyde as a white solid.

Example 402-cyano-3-(3-(3,4,5-trimethoxyphenyl)-1H-indazol-5-yl)acrylamide

To a solution of 3-(3,4,5-trimethoxyphenyl)-1H-indazole-5-carbaldehyde(52.0 mg, 0.16 mmol) and 2-cyanoacetamide (13.2 mg, 0.16 mmol) in THF (1mL) was added DBU (16 μL, 0.16 mmol). The colorless solution becamebright yellow upon addition of DBU, and slowly turned bright red overthe course of 15 minutes. After four hours, the reaction mixture wastaken up in EtOAc (10 mL), and washed with 1M HCl (10 mL), NaHCO₃ (10mL) and brine (10 mL). The organic layer was concentrated and purifiedby preparative TLC, eluting with 9:1 CH₂Cl₂:MeOH, to afford 5.3 mg (9%)of 2-cyano-3-(3-(3,4,5-trimethoxyphenyl)-1H-indazol-5-yl)acrylamide as abright yellow solid. Exact mass: 378.13, M/z found: 379.03 (M+H)⁺.

Example 412-cyano-N-methyl-3-(3-(3,4,5-trimethoxyphenyl)-1H-indazol-5-yl)acrylamide

To a solution of 3-(3,4,5-trimethoxyphenyl)-1H-indazole-5-carbaldehyde(30 mg, 0.100 mmol) and 2-cyano-N-methylacetamide (9.6 mg, 0.100 mmol)in THF (1 mL) was added DBU (10 μL, 0.10 mmol). The colorless solutionbecame bright yellow upon addition of DBU, and slowly turned bright redover the course of 15 minutes. After four hours, the reaction mixturewas taken up in EtOAc (10 mL), and washed with 1M HCl (10 mL), NaHCO₃(10 mL) and brine (10 mL). The organic layer was concentrated andpurified by preparative TLC, eluting with 9:1 CH₂Cl₂:MeOH, to afford 17mg (43%) of2-cyano-N-methyl-3-(3-(3,4,5-trimethoxyphenyl)-1H-indazol-5-yl)acrylamideas a bright yellow solid. Exact mass: 392.15, M/z found: 393.07 (M+H)⁺.

Example 422-cyano-N,N-dimethyl-3-(3-(3,4,5-trimethoxyphenyl)-1H-indazol-5-yl)acrylamide

To a solution of 3-(3,4,5-trimethoxyphenyl)-1H-indazole-5-carbaldehyde(50 mg, 0.16 mmol) and 2-cyano-N-methylacetamide (17.6 mg, 0.16 mmol) inTHF (1 mL) was added DBU (16 μL, 0.16 mmol). The colorless solutionbecame bright yellow upon addition of DBU, and slowly turned bright redover the course of 15 minutes. After four hours, the reaction mixturewas taken up in EtOAc (10 mL), and washed with 1M HCl (10 mL), NaHCO₃(10 mL) and brine (10 mL). The organic layer was concentrated andpurified by preparative TLC, eluting with 9:1 CH₂Cl₂:MeOH, to afford 17mg (26%) of2-cyano-N,N-dimethyl-3-(3-(3,4,5-trimethoxyphenyl)-1H-indazol-5-yl)acrylamideas a bright yellow solid. Exact mass: 406.16, M/z found: 407.05(M+H)⁺.

Example 434-Amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidine-6-carbaldehyde

To a solution of4-Di-tert-butyloxycarbonylamino-7-(3-tert-butyldimethylsiloxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidine-6-carbaldehyde(1.43 g, 2.28 mmol) in dichloromethane (12 mL) was added TFA (5 mL). Thereaction mixture was maintained at ambient temperature for 12 hours,then concentrated under reduced pressure. The residue was redissolved inTHF (12 mL), and 1M aq. HCl (4 mL) was added. The reaction mixture wasstirred at ambient temperature for 24 hours and then diluted with EtOAc(50 mL) and satd. aq. NaHCO₃ (50 mL). The phases were separated and theaqueous phase was extracted with EtOAc (50 mL). The combined organicextracts were washed with brine (50 mL), then concentrated under reducedpressure. The residue was azeotroped with benzene (50 mL) and dried invacuo to afford 0.99 g of the deprotected aldehyde (wet), which was usedwithout further purification.

Example 44 Azetidine (X═H), and 3-hydroxyazetidine (X═OH)cyanoacetamides

Ethylcyanoacetate (1.0 equiv.), azetidine (X═H), or 3-hydroxyazetidine(X═OH) hydrochloride (1.1 equiv.) and triethylamine (1. 5 equiv.) inEtOH (6 mL) were heated at 80° C. for 6 hours. The reaction mixture wasconcentrated and the residue was partitioned between EtOAc (25 mL) andDI water (25 mL). The aqueous phase was extracted with EtOAc (2×30 mL).The combined organic extracts were dried (Na₂SO₄) and concentrated toafford a residue, which was purified by silica gel chromatography (4:1EtOAc/Hexanes for X═H; 16:1 EtOAc/MeOH for X═OH) to afford the desiredcyanoacetamide. Ethylcyanoacetate (550 mg, 4.86 mmol) and azetidinehydrochloride (500 mg) afforded 196.5 mg (33% yield) of azetidinecyanoacetamide. Ethylcyanoacetate (469.4 mg, 4.15 mmol) and3-hydroxyazetidine hydrochloride (500 mg) afforded 89 mg (15% yield) ofazetidine cyanoacetamide.

Example 45 Synthesis of Cyanoacrylamides

General Procedure for Synthesis of Cyanoacrylamide Derivatives:

4-Amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidine-6-carbaldehyde(1.0 equiv.), the appropriate cyanoacetamide (1.2-1.5 equiv.) and DBU(1.5-2.0 equiv.) were stirred in THF (2 mL) at ambient temperature for1-3 days. The reaction mixture was then concentrated and purified bypreparative TLC to afford the cyanoacrylamide as a mixture of (E)- and(Z)-isomers.

Example 45a3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyano-N,N-dimethylacrylamide

Compound prepared from N,N-dimethylcyanoacetamide. See Basheer, A.;Yamataka, H.; Ammal, S. C.; Rappoport, Z. J. Org. Chem. 2007, 72,5297-5312. Yield: 33.8 mg (24%, E:Z=1.7:1). ESI-MS: 405.2 (MH+)

Example 45b3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyano-N-(1-hydroxy-2-methylpropan-2-yl)acrylamide

Compound Prepared from2-cyano-N-(1-hydroxy-2-methylpropan-2-yl)acetamide. See Santilli, A. A.;Osdene, T. S. J. Org. Chem. 1964, 29, 2066-2068. Yield: 17.2 mg (39%),ESI-MS: 449.2 (MH+)

Example 45c3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyano-N,N-diethylacrylamide

Compound prepared from N,N-diethylcyanoacetamide. See Wang, K.; Nguyen,K.; Huang, Y.; Dömling, A. J. Comb. Chem. 2009, 11, 920-927. Yield: 9.2mg (8%), ESI-MS: 433.2 (MH+)

Example 45d3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-(pyrrolidine-1-carbonyl)acrylonitrile

Compound Prepared from N-(2-cyanoacetyl)pyrrolidine. See Wang et al.,Id. Yield: 1.6 mg (3%), ESI-MS: 431.2 (MH+)

Example 45e3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-(azetidine-1-carbonyl)acrylonitrile

Yield: 16.5 mg (32%), ESI-MS: 417.2 (MH⁺).

Example 45f3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-(3-hydroxyazetidine-1-carbonyl)acrylonitrile

Yield: 20.1 mg (38%), ESI-MS: 433.2 (MH⁺).

Example 45g(E)-3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-(4-methylpiperazine-1-carbonyl)acrylonitrile

Compound prepared from N-(2-cyanoacetyl)-N′-methylpiperazine. SeeProenca, F.; Costa, M. Green Chem. 2008, 10, 995-998. Yield: 15.7 mg(25%), ESI-MS: 460.2 (MH+)

Example 463-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyano-N-cyclopropylacrylamide

To a solution of4-tert-butyloxycarbonylamino-7-(3-tert-butyldimethylsiloxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidine-6-carbaldehyde(101 mg, 0.1925 mmol) and N-cyclopropylcyanoacetamide3 (47.8 mg, 2.0equiv.) in THF (2.2 mL) that had been pre-cooled to 0-5° C. was addedDBU (58 μL, 2.0 equiv.). The reaction mixture was allowed to warm toambient temperature and stirred for 1 hour, then maintained at −20° C.for 12 hours. The reaction mixture was concentrated and purified bysilica gel chromatography (2:1 Hexanes/EtOAc) to afford 53.2 mg(E:Z=2:1, 44% yield) of the intermediate protected cyanoacrylamide as ayellow oil. This oil was dissolved in CH₂Cl₂ (3 mL) and TFA (1.5 mL) wasadded. After 16 hours at 20-25° C., the reaction mixture wasconcentrated and the residue was redissolved in THF (6 mL) and 1M aq.HCl (2 mL) was added. The reaction mixture was maintained at ambienttemperature for 8 hours, then quenched with satd. aq. NaHCO3 (20 mL) andbrine (30 mL) and extracted with EtOAc (3×50 mL). The combined EtOAcextracts were dried (MgSO4) and concentrated and the oil afforded waspurified by preparative TLC (3:1 Toluene/IPA, 0.5 cm plate, 2 elutions)to afford the cyanoacrylamide (26 mg, 74% yield over 2 steps) as ayellow oil.

Example 47 1-(4-chlorophenyl)-2-(3-(hydroxymethyl)phenylamino)ethanone

A 100 mL round-bottom flask fitted with a magnetic stir bar was chargedwith 3-aminobenzyl alcohol (1.58 g, 12.85 mmol), potassium carbonate(3.05 g, 22.1 mmol), and N,N′-dimethylformamide (10 mL). The slurry wasstirred while adding 2-bromo-4′-chloroacetophenone (2.85 g, 12.2 mmol)portionwise. The mixture was stirred at room temperature for 2 h. Thereaction was diluted with water (80 mL) and the resulting precipitatewas collected by filtration, washed with water, and dried in vacuo,providing the product (2.89 g, 86% yield).

Example 482-amino-4-(4-chlorophenyl)-1-(3-(hydroxymethyl)phenyl)-1H-pyrrole-3-carbonitrile

A 250 mL round-bottom flask fitted with a magnetic stir bar was chargedwith 1-(4-chlorophenyl)-2-(3-(hydroxymethyl)phenylamino)ethanone (2.88g, 10.4 mmol), potassium hydroxide (85%) (1.8 g, 27 mmol), malononitrile(1.32 g, 20 mmol), water (5 mL), and methanol (50 mL). The mixture wasrefluxed for 1.5 h, during which time a precipitate formed. The mixturewas diluted with water (50 mL) and the precipitate was collected byfiltration, washed with water, and dried in vacuo, providing the product(1.68 g, 50% yield).

Example 49(3-(4-amino-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)methanol

A 250 mL round-bottom flask fitted with a magnetic stir bar was chargedwith2-amino-4-(4-chlorophenyl)-1-(3-(hydroxymethyl)phenyl)-1H-pyrrole-3-carbonitrile(1.60 g, 4.9 mmol), triethyl orthoformate (5 mL), and p-toluenesulfonicacid monohydrate (5 mg). The solution was heated to 100° C. for 45 min.Excess triethyl orthoformate was removed under reduced pressure and theresulting oil was dried briefly in vacuo. To the resulting solid wasadded ammonia (7 M in methanol, 20 mL) and the flask was tightly capped.The solution was stirred at room temperature for 3 h. The solution wasconcentrated under reduced pressure and redissolved in methanol (10 mL).The solution was heated to 80° C. and sodium methoxide (25% w/v inmethanol, 1 mL) was added dropwise. The mixture was refluxed for 2 h,cooled to room temperature and quenched by the addition of water. Thesolution was extracted with ethyl acetate (3×50 mL). The organic layerwas dried over sodium sulfate, filtered, and concentrated under reducedpressure. The resulting oil was purified by Si-gel chromatography (elute1:1 EtOAc/Hex to 100% EtOAc). Intermediate product containing fractionswere combined and concentrated under reduced pressure. The resultingred-brown oil was dissolved in methanol (50 mL) and hydrochloric acid,1M (50 mL) was added. The solution was stirred at room temperature for 1h. The solution was partitioned between ethyl acetate and water andextracted with ethyl acetate. The organic layer was dried over sodiumsulfate, filtered, and concentrated under reduced pressure to yield theproduct as a light yellow solid.

Example 503-(4-amino-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzaldehyde

A 20 mL vial fitted with a magnetic stir bar was charged with(3-(4-amino-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)methanol(145 mg, 0.4 mmol), triethyl amine (0.4 mL, 2.8 mmol), and dimethylsulfoxide (2 mL). A solution of sulfur trioxide-pyridine (200 mg, 1.3mmol) in DMSO (1.2 mL) was added to the vial and the resulting solutionwas stirred for 1 h at room temperature. Hydrochloric acid (1 M, 10 mL)was added and the solution stirred for an additional 10 minutes. Theresulting precipitate was collected by filtration, washed with water,and dried in vacuo, providing the product as a white solid (162 mg, >99%yield).

Example 51(E)-3-(3-(4-amino-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-2-cyanoacrylamide

A 20 mL vial fitted with a magnetic stir bar was charged with3-(4-amino-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzaldehyde(13 mg, 0.04 mmol), 2-cyanoacetamide (4 mg, 0.05 mmol),1,8-diazabicyclo[5.4.0]undec-7-ene (5 mg, 0.03 mmol), andtetrahydrofuran (1 mL). The reaction mixture was heated to 50° C. for 24h. Starting material remained as determined by thin layerchromatography, so piperidinium acetate (5 mg, 0.03 mmol) and 2-propanol(0.5 mL) were added and the solution was heated to 60° C. for anadditional 24 h. The resulting solution was partitioned between diluteNaHCO₃ and EtOAc. The solution was extracted with EtOAc, and the organiclayer was dried over sodium sulfate, filtered, and concentrated underreduced pressure. The resulting residue was purified by Si-gelchromatography (elute 3:1 EtOAc/Hex to 100% EtOAc) to yield the productas a white solid (2 mg, 13% yield). Exact Mass: 414.10. M/z found: 415.1(M+H)⁺

Example 52(E)-3-(3-(4-amino-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-2-cyano-N,N-dimethylayerylamide

A 20 mL vial fitted with a magnetic stir bar was charged with3-(4-amino-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzaldehyde(35 mg, 0.10 mmol), N,N′-dimethyl-2-cyanoacetamide (12 mg, 0.11 mmol),1,8-diazabicyclo[5.4.0]undec-7-ene (5 mg, 0.03 mmol), and 2-propanol (1mL). The reaction mixture was heated to 60° C. for 24 h. Only startingmaterial was present as determined by thin layer chromatography, so thesolution was heated to 80° C. for an additional 24 h. The solution wasconcentrated under reduced pressure and the resulting residue wasredissolved in DMSO (0.8 mL) and the product was purified by RP-HPLC(gradient: 20-95% MeCN/water with 0.1% TFA over 25 min, retention time˜6.8 min). Product containing fractions were combined and the solventremoved under reduced pressure and the resulting solid was purified bySi-gel chromatography (elute with EtOAc). Collect the product as a whitesolid (6.1 mg, 14% yield). Exact Mass: 442.13. M/z found: 443.0 (M+H)⁺.

Example 53(E)-3-(3-(4-amino-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-2-(azetidine-1-carbonyl)acrylonitrile

A 20 mL vial fitted with a magnetic stir bar was charged with3-(4-amino-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzaldehyde(15 mg, 0.04 mmol), 3-(azetidin-1-yl)-3-oxopropanenitrile (12 mg, 0.1mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (5 mg, 0.03 mmol), and2-propanol (1 mL). The reaction mixture was heated to 60° C. for 24 h.The solution was concentrated under reduced pressure and the resultingresidue was purified by Si-gel chromatography (elute 100% EtOAc to 10%MeOH/EtOAc). Collect the product as a white solid (16 mg, 82% yield).Exact Mass: 454.13. M/z found: 455.0 (M+H)⁺.

Example 54(E)-3-(3-(4-amino-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-2-(pyrrolidine-1-carbonyl)acrylonitrile

A 20 mL vial fitted with a magnetic stir bar was charged with3-(4-amino-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzaldehyde(15 mg, 0.04 mmol), 3-oxo-3-(pyrrolidin-1-yl)propanenitrile (14 mg, 0.1mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (5 mg, 0.03 mmol), and2-propanol (1 mL). The reaction mixture was heated to 60° C. for 24 h.The solution was concentrated under reduced pressure and the resultingresidue was purified by Si-gel chromatography (elute 100% EtOAc to 10%MeOH/EtOAc). Collect the product as a white solid (12 mg, 59% yield).Exact Mass: 468.15. M/z found: 469.1 (M+H)⁺.

Example 55(E)-3-(3-(4-amino-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-2-(3-hydroxyazetidine-1-carbonyl)acrylonitrile

A 20 mL vial fitted with a magnetic stir bar was charged with3-(4-amino-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzaldehyde(15 mg, 0.04 mmol), 3-(3-hydroxyazetidin-1-yl)-3-oxopropanenitrile (14mg, 0.1 mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (5 mg, 0.03 mmol), and2-propanol (1 mL). The reaction mixture was heated to 60° C. for 24 h.The solution was concentrated under reduced pressure and the resultingresidue was purified by Si-gel chromatography (elute 100% EtOAc to 10%MeOH/EtOAc). The product was collected as a white solid (13 mg, 64%yield). Exact Mass: 470.13. M/z found: 471.0 (M+H)⁺.

Example 56(E)-3-(3-(4-amino-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-2-(4-methylpiperazine-1-carbonyl)acrylonitrile

A 20 mL vial fitted with a magnetic stir bar was charged with3-(4-amino-5-(4-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzaldehyde(16 mg, 0.05 mmol), 3-(4-methylpiperazin-1-yl)-3-oxopropanenitrile (17mg, 0.11 mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (5 mg, 0.03 mmol),and 2-propanol (1 mL). The reaction mixture was heated to 60° C. for 24h. Only starting material was present as determined by thin layerchromatography, so the solution was heated to 70° C. for an additional24 h. The solution was concentrated under reduced pressure and theresulting residue was redissolved in DMSO (0.8 mL) and the product waspurified by RP-HPLC (gradient: 5-80% MeCN/water with 0.1% TFA over 25min). Collect the product as a white solid (3 mg, 13% yield). ExactMass: 497.17. M/z found: 498.1 (M+H)⁺

Example 57 2-(3-(hydroxymethyl)phenylamino)-1-(4-phenoxyphenyl)ethanone

A 100 mL round-bottom flask fitted with a magnetic stir bar was chargedwith 3-aminobenzyl alcohol (1.3 g, 10.6 mmol), potassium carbonate (1.4g, 10.1 mmol), and N,N′-dimethylformamide (15 mL). The slurry wasstirred while adding 2-bromo-4′-chloroacetophenone (3.04 g, 10.4 mmol)portionwise. The mixture was heated to 50° C. and stirred for 2 h. Thereaction mixture was partitioned between EtOAc and water, and thenextracted with EtOAc. The organic layers were combined, dried oversodium sulfate, filtered, and concentrated under reduced pressure. Theresulting residue was purified by Si-gel chromatography (elute 1:3EtOAc/hexanes to 1:1 EtOAc/hexanes). The product was collected as awhite solid (1.39 g, 40% yield).

Example 582-amino-1-(3-(hydroxymethyl)phenyl)-4-(4-phenoxyphenyl)-1H-pyrrole-3-carbonitrile

A round-bottom flask fitted with a magnetic stir bar was charged with2-(3-(hydroxymethyl)phenylamino)-1-(4-phenoxyphenyl)ethanone (1.39 g,4.17 mmol), potassium hydroxide (85%) (0.8 g, 12 mmol) dissolved inwater (3 mL), malononitrile (0.50 g, 7.6 mmol), and methanol (15 mL).The mixture was heated to 80° C. for 2 h. The reaction mixture waspartitioned between EtOAc and water, and then extracted with EtOAc. Theorganic layers were combined, dried over sodium sulfate, filtered, andconcentrated under reduced pressure. The resulting residue was purifiedby Si-gel chromatography, providing the product (0.91 g, 57% yield).Exact Mass: 381.15. M/z found: 382.1 (M+H)⁺

Example 59(3-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)methanol

A 100 mL round-bottom flask fitted with a magnetic stir bar was chargedwith2-amino-1-(3-(hydroxymethyl)phenyl)-4-(4-phenoxyphenyl)-1H-pyrrole-3-carbonitrile2-amino-4-(4-chlorophenyl)-1-(3-(hydroxymethyl)phenyl)-1H-pyrrole-3-carbonitrile(0.91 g, 2.7 mmol), triethyl orthoformate (3 mL), and p-toluenesulfonicacid monohydrate (10 mg). The solution was heated to 100° C. for 1.5 h.The reaction mixture was partitioned between EtOAc and water. Theorganic layer was washed with 5% NaHCO₃ and then dried over sodiumsulfate, filtered, and concentrated under reduced pressure. Theresulting residue was purified by Si-gel chromatography (elute 20% to40% EtOAc/Hex). The resulting oil was concentrated under reducedpressure and redissolved in ammonia/methanol solution (7 M, 10 mL) andthe flask was tightly capped and stirred at room temperature over night.The reaction mixture was concentrated under reduced pressure andredissolved ammonia/methanol solution (7 M, 10 mL), the flask tightlycapped and stirred at room temperature for an additional 2 h. Thesolution was concentrated under reduced pressure and redissolved inmethanol (20 mL) and sodium methoxide (25% w/v in methanol, 1 mL) wasadded. The mixture was refluxed for 2 h, cooled to room temperature andquenched by the addition of hydrochloric acid (1M, 10 mL). The solutionwas stirred for 1 h at room temperature. The reaction solution wasneutralized by the addition of sodium bicarbonate and then was extractedwith ethyl acetate (3×50 mL). The organic layer was dried over sodiumsulfate, filtered, and concentrated under reduced pressure. Theresulting oil was purified by Si-gel chromatography (elute 100% EtOAc to10% MeOH/EtOAc) to yield the product as a light yellow solid (0.38 g,38% yield).

Example 603-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzaldehyde

A 20 mL vial fitted with a magnetic stir bar was charged with(3-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)methanol(207 mg, 0.51 mmol), triethyl amine (0.5 mL, 3.6 mmol), and dimethylsulfoxide (2.5 mL). A solution of sulfur trioxide pyridine (245 mg, 1.6mmol) in DMSO (1.5 mL) was added to the vial and the resulting solutionwas stirred for 1 h at room temperature. Hydrochloric acid (1 M, 5 mL)was added and the solution stirred for an additional 5 minutes. Theresulting solution was partitioned between EtOAc and water and theorganic layer was washed with hydrochloric acid (1 M, 2×), sodiumbicarbonate (5%, 1×), water (3×), and brine (1×). The organic layer wasthen dried over sodium sulfate, filtered, and concentrated under reducedpressure. The resulting residue was purified by Si-gel chromatography(elute 100% EtOAc to 10% MeOH/EtOAc), providing the product as a whitesolid (141 mg, 68% yield). Exact Mass: 406.14. M/z found: 407.1 (M+H)⁺.

Example 61 General methods for the synthesis of(E)-3-(3-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-2-cyano-acrylamidesExample 61a(E)-3-(3-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-2-cyano-N,N-dimethylayerylamide

A 20 mL vial fitted with a magnetic stir bar was charged with3-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzaldehyde(20 mg, 0.05 mmol), the appropriate cyanoacetamide (0.05 mmol),1,8-diazabicyclo[5.4.0]undec-7-ene (5 mg, 0.03 mmol), and 2-propanol (1mL). The reaction mixture was heated to 60° C. for 18 h. The solutionwas concentrated under reduced pressure and the resulting residue wasredissolved in DMSO (0.8 mL) and the product was purified by RP-HPLC(gradient: 20-95% MeCN/water with 0.1% TFA over 25 min). The product isa white solid (4.2 mg, 17% yield). Exact Mass: 500.20. M/z found: 501.3(M+H)⁺.

Example 61b(E)-3-(3-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-2-(4-methylpiperazine-1-carbonyl)acrylonitrile

Employing the procedure described above, the named compound wassynthesized. The compound is a white solid (9.2 mg, 34% yield). ExactMass: 555.24. M/z found: 556.1 (M+H)⁺.

Example 61c(E)-3-(3-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-2-(3-hydroxyazetidine-1-carbonyl)acrylonitrile

Employing the procedure described above, the named compound wassynthesized. The product is a white solid (9.7 mg, 37% yield). ExactMass: 528.19. M/z found: 529.2 (M+H)⁺.

Example 61d(E)-3-(3-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-2-cyanoacrylamide

Employing the procedure described above, the named compound wassynthesized. The compound is a light yellow solid (6.8 mg, 29% yield).Exact Mass: 472.16. M/z found: 473.0 (M+H)⁺.

Example 62 2-cyano-N-(2-hydroxyethyl)acetamide

A 100 mL round-bottom flask fitted with a magnetic stir bar was chargedwith methyl cyanoacetate (4.94 g, 49.9 mmol), and ethanolamine (3.01 g,49.8 mmol). The reaction mixture was stirred for 2 h at roomtemperature, at which point the solution was concentrated under reducedpressure and dried in vacuo to yield the product as a white solid (6.91g, >99% yield).

Example 63(E)-3-(3-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-2-cyano-N-(2-hydroxyethyl)acrylamide

A 4 mL vial fitted with a magnetic stir bar was charged with3-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzaldehyde(9 mg, 0.02 mmol), 2-cyano-N-(2-hydroxyethyl)acetamide (4 mg, 0.03mmol), piperidinium acetate (1 mg, 0.01 mmol), and 2-propanol (0.5 mL).The reaction mixture was heated to 60° C. for 24 h. The solution wasconcentrated under reduced pressure and the resulting residue waspurified by Si-gel chromatography (elute 100% EtOAc to 10% MeOH/EtOAc)to yield the product, which was further purified by RP-HPLC (gradient:20-95% MeCN/water with 0.1% TFA over 25 min) to yield the product as awhite solid (3.1 mg, 27% yield). Exact Mass: 516.19. M/z found: 517.0(M+H)⁺.

Example 64 Determination of Inhibitory Constants

Methods. RSK2 CTD and His6-ERK2 were expressed and purified as described(Cohen et al., Science, 308: 1318). The C436V mutant of RSK2 CTD wasgenerated by Quikchange mutagenesis (Stratagene) and wasindistinguishable from WT RSK2 CTD in kinase activity assays, asdescribed previously. WT and C436V RSK2 CTD (10 μM) were activated byHis6-ERK2 (10 μM) in 20 mM HEPES [pH 8.0], 10 mM MgCl₂, 2.5 mMtris(2-carboxyethyl)phosphine (TCEP), 0.2 mg/mL BSA and 200 μM ATP for30 min at 22° C. Activated RSK2 CTD (5 nM) in 20 mM HEPES [pH 8.0], 10mM MgCl₂, 2.5 mM tris(2-carboxyethyl)phosphine (TCEP), 0.25 mg/mL BSA,and 100 μM ATP were pre-incubated with inhibitors (ten concentrations,in duplicate) for 30 min. Kinase reactions were initiated by theaddition of 5 μCi of [γ-³²P]ATP (6000 Ci/mmol, NEN) and 167 μM peptidesubstrate (RRQLFRGFSFVAK) (SEQ ID NO:1) and performed for 30 min at roomtemperature. Kinase activity was determined by spotting 5 μL of eachreaction onto dried sheets of nitrocellulose that had been pre-washedwith 1 M NaCl in 0.1% H₃PO₄. The nitrocellulose sheets were washed oncewith 1% AcOH solution and twice with a solution of 1 M NaCl in 0.1%H₃PO₄ (5-10 min per wash). Dried blots were exposed for 30 min to astorage phosphor screen and scanned by a Typhoon imager (GE LifeSciences). Data were quantified using the SPOT program (Knight, Z. etal. Nature Protocols, 2: 2459-66), and IC₅₀ values were determined usingGraphPad Prism 4.0 software.

Results.

Table 1 provides half-maximal inhibitory concentrations (IC₅₀ in μM) forelectrophilic pyrrolo[2,3-d]pyrimidines 1-8 toward WT RSK2 and C436VRSK2 C-terminal kinase domain (CTD). Compounds 4-6 were additionallytested for RSK2 CTD inhibition in the presence of 10 mM reducedglutathione (GSH). Despite reacting reversibly withcyanoacrylates/cyanoacrylamides 4-6 (GSH reaction with compounds 4-6 wasmonitored by UV/visible spectroscopy at 350-400 nm) and being present atone million times the concentration of RSK2 CTD, glutathione had noeffect on the inhibitory potency of 4-6. Consistent with the formationof a covalent adduct between Cys436 and the electrophilic beta-carbon ofthe cyanoacrylate and cyanoacrylamide moieties of 4-8, mutation ofcysteine-436 to valine (C436V) resulted in a>1000-fold loss ininhibitory potency. Finally, cyanoacrylates and cyanoacrylamides 4-8were significantly more potent than compounds I-3, which are based onmore conventional Michael acceptors (vinyl ketone, acrylate ester,acrylonitrile). N/d, not determined.

TABLE 1 IC₅₀ value for selected compounds and RSK2 species. IC₅₀ (μM) WTRSK2 + WT RSK2 C436V RSK2 10 mM GSH Me enone (1) 0.087 1.7 n/d Meacrylate (2) 0.25 0.19 n/d acrylonitrile (3) 0.75 1.5 n/d CN—OMe (4)0.013 >10 0.015 CN—OtBu (5) 0.007 >10 0.006 CN—NH2 (6) 0.003 5 0.004CN—NHiPr (7) 0.005 5.5 n/d CN—NHBn (8) 0.040 n/d n/d

Example 65 Mass Spectrometry of RSK2 CTD Reaction with Compounds 1-8

Methods.

RSK2 CTD (human RSK2, 399-740) was expressed in E. coli as a His₆-taggedfusion protein and purified by Ni/NTA affinity chromatography, followedby cleavage of the His₆-tag and further purification by size exclusionchromatography. RSK2 CTD (5 μM) was incubated with the indicatedcompounds (25 μM, equiv) for 1 h at room temperature in buffer (20 mMHEPES [pH 8.0], 100 mM NaCl, 10 mM MgCl₂). The reaction was stopped byadding an equal volume of 0.4% formic acid, and the samples wereanalyzed by liquid chromatography (Microtrap C18 Protein column [MichromBioresources], 5% MeCN, 0.2% formic acid, 0.25 mL/min; eluted with 95%MeCN, 0.2% formic acid) and in-line ESI mass spectrometry (LCT Premier,Waters). Molecular masses of RSK2 CTD and electrophilicpyrrolo[2,3-d]pyrimidine adducts were determined with MassLynxdeconvolution software and are shown in histogram format.

Results.

As depicted in FIG. 1A and FIG. 1B, despite their higher potency as RSK2inhibitors, cyanoacrylates and cyanoacrylamides 4-8 do NOT irreversiblymodify the RSK2 C-terminal kinase domain (CTD), as revealed byhigh-resolution mass spectrometry analysis. Pyrrolo[2,3-d]pyrimidines1-3 contain conventional electrophilic “warheads” and, as expected,formed irreversible 1:1 adducts with RSK2. This conclusion is supportedby the formation of a new peak in the mass spectrum corresponding to themolecular mass of RSK2 CTD plus the molecular mass of the electrophiliccompound (FIG. 1A). Note that modification of RSK2 CTD by acrylate 2 andacrylonitrile 3 was somewhat slower relative to enone 1, due to thelower intrinsic electrophilicity of the acrylate/acrylonitrile warheads.

In contrast to compounds I-3, the cyanoacrylate and cyanoacrylamideinhibitors 4-8 did not form irreversible adducts with RSK2 CTD, as shownby the presence of a single peak in the mass spectra corresponding tothe unmodified RSK2 CTD. Despite the lack of irreversible RSK2modification, compounds 4-8 are significantly more potent inhibitors ofRSK2 kinase activity than the irreversible inhibitors 1-3 (Table 1). TheCys436Val mutant of RSK2 (C436V) was ˜1000-fold less sensitive tocompounds 4-8 (Table 1), demonstrating that potent inhibition requiresCys436. Together, these data suggest that compounds 4-8, all of whichcontain a cyanoacrylate or cyanoacrylamide electrophile, inhibit RSK2kinase activity by forming a reversible covalent bond between theelectrophilic beta-carbon of the cyanoacrylate/cyanoacrylamide moietyand Cys436 of RSK2. Additional evidence for a reversible covalentmechanism of inhibition for this class of electrophiles (cyanoacrylatesand cyanoacrylamides) is provided below.

Example 66 Recovery of RSK2 CTD Activity Upon Dialysis

Methods.

The indicated pyrrolo[2,3-d]pyrimidines (1 μM) were added to a solutionof RSK2 CTD (50 nM, pre-activated with 1 equiv of ERK2) in a buffercontaining 20 mM HEPES [pH 8.0], 10 mM MgCl₂, 2.5 mMtris(2-carboxyethyl)phosphine (TCEP), 0.25 mg/mL BSA, and 100 μM ATP.After 60 min at rt, the reactions were transferred to a dialysiscassette (0.1-0.5 mL Slide-A-Lyzer, MWCO 10 kDa, Pierce) and dialyzedagainst 2 L of buffer (20 mM Hepes [pH 8.0], 10 mM MgCl₂, 1 mM DTT) at4° C. The dialysis buffer was exchanged after 2 h, and then wasexchanged every 24 h until the end of the experiment. Aliquots wereremoved from the dialysis cassettes every 24 h, flash frozen in liquidnitrogen, and subsequently analyzed for RSK2 kinase activity intriplicate. Kinase activity for each sample was normalized to the DMSOcontrol for that time point and expressed as the mean±SD.

Results.

As depicted in FIG. 2, RSK2 CTD kinase activity recovers from inhibitionby pyrrolo[2,3-d]pyrimidines 6 and 7 upon dialysis, indicating that 6and 7 are reversible inhibitors. The data in FIG. 2 show that, uponextensive dialysis at 4° C., RSK2 kinase activity recovers in atime-dependent manner from inhibition by an excess (20 equiv, 1.0 μM) ofcyanoacrylamides 6 (˜60% recovery) and 7 (˜25% recovery). Thus,cyanoacrylamides 6 and 7 are slowly dissociating, reversible inhibitorsof RSK2 with dissociation half-times of ˜3 days and >4 days,respectively, under these conditions (dialysis at 4° C.). Note thatdissociation is more rapid at room temperature and proceeds tocompletion in the presence of an irreversible competitor (see FIG. 3).In contrast to the partial recovery of kinase activity observed withcyanoacrylamides 6 and 7, RSK2 CTD remained completely inhibited byenone 1 and fluoromethylketone 9 during 4 days of dialysis, furtherdemonstrating that these compounds are irreversible inhibitors. Theseresults are consistent with the LCMS data, which show that enone 1 (FIG.1A) and fluoromethylketone 9 (FIG. 3) react irreversibly with RSK2,whereas cyanoacrylates and cyanoacrylamides do not form irreversibleRSK2 adducts. Further evidence that cyanoacrylate- andcyanoacrylamide-substituted pyrrolo[2,3-d]pyrimidines form slowlydissociating, fully reversible complexes with RSK2 is provided in FIG.3.

Example 67 Dissociation Kinetics of Reversible Covalent Inhibitors

Methods.

RSK2 CTD (5 μM) in 20 mM HEPES [pH 8.0], 10 mM MgCl₂, 100 mM NaCl, 2.5mM tris(2-carboxyethyl)phosphine (TCEP) and 0.2 mg/mL BSA waspreincubated with 10 μM inhibitor 1, 4-7 (or DMSO control) for 60 min atroom temperature. FMK 9 (100 μM) was then added, and aliquots wereremoved at different time points (0.5-1500 min) and immediately quenchedby mixing with an equal volume of 0.4% formic acid. Samples wereanalyzed by LCMS with an LCT Premier mass spectrometer and MassLynxdeconvolution software, as described in FIG. 1. Peaks corresponding toempty RSK2 CTD (in the case of pre-treatment with 4-7; pre-treatmentwith enone 1 produced the expected mass shift, which did not changeafter FMK addition) and FMK-modified RSK2 CTD were integrated at eachtime point, and the percent FMK adduct plotted as a function of time(denoted “% Dissociation” in the graph on the left side of FIG. 3). Thegraph on the right side of FIG. 3 shows FMK labeling kinetics in theabsence of any competitor. Kinetic data (% FMK adduct vs. time) were fitto a single exponential (PRISM 4.0) to obtain dissociation half-timesdepicted in the table. Control experiments showed that C436V RSK2 wasnot modified by FMK 9 under these conditions.

Results.

As depicted in FIG. 3, cyanoacrylates/cyanoacrylamides 4-7 dissociatewith a half-time of hours from intact, folded RSK2 CTD, as measured bycompetitive labeling with fluoromethylketone 9. A large molar excess offluoromethylketone 9 (100 μM, 20 equiv; “FMK” in FIG. 3) rapidly andirreversibly modified RSK2 CTD (t_(1/2)<2 min), as revealed by LCMSanalysis of the RSK2 CTD (FIG. 3, upper left graph). By contrast, whenRSK2 CTD was first treated with cyanoacrylates/cyanoacrylamides 4-7 (2.0equiv), modification resulting from subsequent treatment with FMK 9 (100μM, 20 equiv) was much slower, occurring with a half-time (t_(1/2)) of42-245 min (FIG. 3, upper right graph). Because modification of apo-RSK2CTD by FMK 9 occurs in less than 2 min, the observed FMK modificationrate of RSK2 pre-bound to compounds 4-7 is approximately equal to thedissociation rate of the pre-bound cyanoacrylate/cyanoacrylamide. Methylvinyl ketone 1, a conventional Michael acceptor, did not dissociateunder these conditions, and FMK labeling was not observed, even after 24h (FIG. 3). These data are consistent with the dialysis experiments inFIG. 2 and reveal that cyanoacrylates/cyanoacrylamides 4-7 dissociateslowly, yet completely, from the intact, functional RSK2 CTD, withdissociation half-times of 1-4 h. The data further reveal that the“off-rates” of the cyanoacrylates/cyanoacrylamide inhibitors can betuned by modifying the ester and amide substituents. The N-isopropylcyanoacrylamide had the slowest off-rate, consistent with potent RSK2inhibitory activity in vitro and in cell-based assays (see Table 1 andFIG. 6).

Example 68 Covalent Bond Formation Between Cys436 and Cmpd 7

Methods.

To a solution of cyanoacrylamide 7 (1.0 equiv, 100-200 μM) inphosphate-buffered saline (PBS) was added WT RSK2 CTD, C436V RSK2 CTD(1.5 equiv), or buffer alone. After 10 min at rt, the UV/Visibleabsorbance spectrum was acquired (NanoDrop 1000 Spectrophotometer). SDS(final concentration of 2%), guanidine (final concentration 3 M, pH 8),or proteinase K (0.02 equiv based on RSK2 CTD) was added and theUV/Visible absorbance spectrum was recorded after 1 min at RT (SDS,guanidinium HCl) or 3 h at 37° C. (proteinase K). Spectra from theproteinase K and SDS addition experiments are shown in the middle andlower panels, respectively. In each experiment, absorbance values at 400nm were normalized to the value of cyanoacrylamide 7 in buffer alone andplotted on the bar graph. Control experiments showed that neither SDS,guanidine, nor proteinase K affected the absorbance spectrum ofcyanoacrylamide 7 at 400 nm.

Results.

As depicted in FIG. 4, the covalent bond between Cys436 andcyanoacrylamide 7 reverses within seconds upon denaturation orproteolytic digestion of RSK2. We monitored covalent bond formation andreversal between RSK2 and cyanoacrylamide 7 by UV/visible absorptionspectroscopy. Cyanoacrylamide 7 absorbs visible light with a peak at˜400 nm (FIG. 4, middle and lower panels), as expected for acyanoacrylamide moiety conjugated to a heteroaromatic system. Additionof excess RSK2 CTD (1.5 equiv) to cyanoacrylamide 7 resulted in completedisappearance of the 400 nm peak, indicating disruption of thecyanoacrylamide chromophore by nucleophilic attack of Cys436. Bycontrast, addition of RSK2 CTD carrying the C436V mutation had no effecton the absorbance spectrum of cyanoacrylamide 7 (FIG. 4, bar graph).This control further substantiates our interpretation that loss of the400 nm peak results from formation of a covalent bond between Cys436 ofRSK2 and the electrophilic beta-carbon of the cyanoacrylamide moiety ofcompound 7.

We used three independent methods to disrupt the folded state of theRSK2 kinase domain bound to cyanoacrylamide 7: (1) 0.02 equiv proteinaseK (“Prot K”), a non-specific proteinase that digests folded proteinsinto small peptides, (2) 2% sodium dodecyl sulfate (“SDS”), awell-characterized protein-denaturing detergent, and (3) 3 M guanidineHCl (“Guan”), a chaotrope that disrupts the native three-dimensionalfold of most proteins. All three protein denaturants resulted in thereappearance of the 400 nm absorbance peak, indicating that the covalentbond between Cys436 and cyanoacrylamide 7 had broken. Covalent bondreversal occurred within seconds, concomitant with RSK2 denaturation (bySDS and guanidine HCl) or proteolytic digestion (complete digestion with0.02 equiv proteinase K proceeded over ˜3 h). Absorbance spectra areshown in the middle and lower panels for experiments with proteinase Kand SDS, respectively (absolute absorbance values are different in thetwo experiments because different concentrations of cyanoacrylamide 7were used). Absorbance values for all three conditions were normalizedto the absorbance of cyanoacrylamide 7 in buffer alone and are shown inthe bar graph. FIG. 5 shows LCMS chromatograms derived from similarexperiments, proving that the addition of protein denaturants to thecovalent complex formed between cyanoacrylamide 7 and RSK2 CTD resultsin the quantitative liberation of cyanoacrylamide 7.

Our data suggest that cyanoacrylamide inhibitors are unlikely to formpermanent covalent adducts with cellular proteins, because once theprotein is unfolded and/or proteolytically digested (a likelypre-condition for eliciting a delayed immune reaction), the covalentbond becomes kinetically and thermodynamically unstable and rapidlydissociates. We demonstrated this behavior with a high-affinity(nanomolar to picomolar) complex between cyanoacrylamide 7 and RSK2 CTD,in which the dissociation half-time changed from ˜3 h in the foldedstate to less than 1 min upon denaturation of the kinase domain.Consistent with these observations, glutathione, an abundantcysteine-containing peptide, formed covalent adducts with compounds 4-8(shown by UV/Visible spectroscopy) that rapidly dissociated, as shown by(1) rapid recovery of the cyanoacrylate/cyanoacrylamide chromophore upondilution, and (2) unperturbed RSK2 inhibitory potency in the presence of10 mM glutathione (Table 1). The dependence of the rate of covalent bonddissociation of a protein thiol/electrophile adduct on the folded stateof the protein has not, to our knowledge, been described.

Example 69 Recovery of Cmpd 7 after Denaturation of RSK2 CTD/Cmpd 7Complex

Methods. Cyanoacrylamide 7 (250 μM) was incubated in the absence orpresence of RSK2 CTD (300 μM) for 10 min in a total volume of 50 μL.Guanidine hydrochloride (50 μL, 6 M, pH 8) was added and the contentswere mixed gently for 1 min, after which acetonitrile was added to afinal concentration of 50%. The solution was filtered (0.2 μm pore size)and analyzed by LCMS (20 μL injection, Waters XTerra MS C18 column,5-70% MeCN/water+0.1% formic acid over 20 min; Waters 2695 AllianceSeparations Module, Waters Micromass ZQ mass spectrometer).

Results.

As depicted in FIG. 5A and FIG. 5B, consistent with the spectroscopicdata shown in FIG. 4, LCMS analysis revealed quantitative recovery ofcyanoacrylamide 7 after denaturation of the RSK2 CTD/cyanoacrylamide 7complex with 3 M guanidine. The first chromatogram (FIG. 5A) (λ=350 nm)shows cyanoacrylamide 7 dissolved in buffer with 3 M guanidine HCl. Thesecond chromatogram (FIG. 5B)(λ=350 nm) shows recovery of purecyanoacrylamide 7 after treatment of the RSK2 CTD/cyanoacrylamide 7complex with 3 M guanidine HCl. Two major peaks, corresponding to E- andZ-isomers of cyanoacrylamide 7, respectively, were observed in bothsamples. Peak areas were similar in the control and RSK2 CTD-treatedsamples, indicating quantitative recovery of cyanoacrylamide 7 afterdenaturation with guanidine. MS analysis of each peak confirmed itsidentity as E- or Z-cyanoacrylamide 7 (calculated MW, 418.2; observed[M+H], 419.1).

Example 70 Inhibition of RSK2 Autophosphorylation of Ser386

Methods.

HEK-293 cells in a 75 cm² flask (˜80% confluent) were transfected withthe pMT2 expression vector encoding HA-tagged RSK2 using Lipofectamine2000 (Invitrogen) according to the manufacturer's protocol. After 12 h,the cells were trypsinized and seeded into 6-well plates at 600,000cells per well in DMEM with 10% serum. After an additional 16 h, thecells were deprived of serum for 4 h and then treated with the indicatedconcentrations of inhibitors for 2 h in serum-free DMEM. Followinginhibitor treatment, the cells were stimulated for 30 min with phorbolmyristate acetate (PMA) (100 ng/ml), then washed with 2 mL cold PBS andfrozen onto the plate at −80° C. The cells were thawed and scraped into80 μL of lysis buffer (20 mM Hepes pH 7.9, 450 mM NaCl, 25% glycerol, 3mM MgCl₂, 0.5 mM EDTA) with protease (Complete, Roche) and phosphatase(Cocktails 1 and 2, Sigma-Aldrich) inhibitors. The lysates were clearedby centrifugation and normalized via Bradford assay quantification.Laemmli sample buffer was added to the lysates, and the proteins wereseparated by 10% SDS-PAGE and analyzed by Western blot withphospho-Ser386 RSK2 (1:500 dilution, rabbit mAb, Cell Signaling #9335)and anti-HA (1:1000 dilution, 12CA5 mouse monoclonal, Roche) antibodies.Images were recorded on a LI-COR Odyssey imaging system (LI-CORBiosciences), and band intensities were integrated using LI-CORsoftware. For each condition, the ratio of the phospho-Ser386 signal tothe HA-RSK2 signal was calculated and expressed as a percentage of theDMSO control value (+PMA). These data are presented in the graph.

Results.

As depicted in FIG. 6, cyanoacrylate 5 and cyanoacrylamides 6 and 7inhibit RSK2 autophosphorylation of Ser386 in mammalian cells. Note thatthe N-isopropyl cyanoacrylamide 7 was the most potent (IC₅₀<30 nM) amongall the inhibitors tested, whereas cyanoacrylamide 6 had weak activity(IC₅₀˜1000 nM). The data show that the cyanoacrylate and cyanoacrylamideinhibitors are cell permeable and sufficiently potent to compete withhigh intracellular concentrations of ATP and glutathione.

Example 71 Determination of General Utility of Activated Olefins forInhibition of Therapeutically Relevant Proteins

Methods

For RSK2 kinase assays, see Example 64 above. Full-length human T175ANEK2 (referred to as “NEK2”) was expressed and purified as previouslydescribed (Knapp S. et al. J. Biol. Chem., 2007, 282: 6833-6842). TheC22V mutant of NEK2 was generated by Quikchange mutagenesis (Stratagene)and was indistinguishable from NEK2 in kinase activity assays. NEK2kinases (60 nM) in 20 mM Hepes, pH 7.6, 10 mM MgCl₂, 1 mM EDTA, 0.2mg/mL BSA, and 100 μM ATP were pre-incubated with inhibitors (8-10concentrations, in duplicate) for 30 min at room temperature. Kinasereactions were initiated by the addition of 0.4 μCi/μL of γ-³²P-ATP(6000 Ci/mmol, NEN) and 2.37 mg/mL β-casein (Sigma) and incubated for 30minutes at room temperature.

Human PLK1 (Millipore, catalog number 14-777M) (7.2 nM) in 20 mM Hepes,pH 7.6, 10 mM MgCl₂, 1 mM EDTA, 0.2 mg/mL BSA, and 100 μM ATP waspre-incubated with inhibitors (8-10 concentrations, in duplicate) for 30min at room temperature. Kinase reactions were initiated by the additionof 0.4 μCi/μL of γ-³²P-ATP (6000 Ci/mmol, NEN) and 0.5 mg/mLdephosphorylated α-casein (Sigma) and incubated for 30 min at roomtemperature. Kinase activity was determined by spotting 5 μL of eachreaction onto dried sheets of nitrocellulose pre-washed with 1M NaCl in0.1% H₃PO₄. After blotting each kinase reaction, the nitrocellulosesheets were washed once with 1% AcOH solution. Kinase activity wasdetermined by spotting 5 μL of each reaction onto dried sheets ofnitrocellulose that had been pre-washed with 1 M NaCl in 0.1% H₃PO₄. Thenitrocellulose sheets were washed once with 1% AcOH solution and twicewith a solution of 1 M NaCl in 0.1% H₃PO₄ (5-10 minutes per wash). Driedblots were exposed for 30 minutes to a storage phosphor screen andscanned by a Typhoon imager (GE Life Sciences). Data were quantifiedusing the SPOT program (Knight, Z. et al. Nature Protocols, 2: 2459-66),and IC₅₀ values were determined using GraphPad Prism 4.0 software.

Results.

To test the general utility of activated olefins (“Michael acceptors”),including cyanoacrylamides, as cysteine-targeting moieties for theinhibition of therapeutically relevant proteins, we synthesized a panelof cyanoacrylamides that were substituted with heterocycles commonlyfound in kinase inhibitor drugs (e.g., azaindoles, indazoles, pyridines,pyrazoles, biaryls). We also synthesized acrylonitriles that were eitherunsubstituted on the nitrile-bearing carbon (Table 2a, entries 28 and30) or were substituted with non-carboxamide electron withdrawing groupson the nitrile-bearing carbon (Table 2a, entries 19-21, 31, 32). Thesenovel compounds were evaluated for their ability to inhibit thefollowing kinases in vitro: WT RSK2, C436V RSK2, WT NEK2, C22V NEK2,and/or PLK1. Note that the wild-type versions of these kinases all havea cysteine in a similar location of the ATP binding site, immediatelyC-terminal to the “glycine-rich” loop.

From these data, we conclude: (1) changing the structure of theheterocycle dramatically affects the kinase inhibitory potency andselectivity of the cyanoacrylamides and acrylonitriles. Thus, even theserelatively simple “fragments” (MW<300) show steep structure-activityrelationships. (2) Acrylonitriles that are substituted with a secondelectron withdrawing group (e.g., carboxamide) on the nitrile-bearingcarbon are more potent than simple acrylonitriles containing hydrogen onthe nitrile-bearing carbon (compare entries 30 vs. 31 and 22 in Table2a; entries 3 vs. 4-8 in Table 1). (3) Where tested, the Cys to Valmutants of RSK2 and NEK2 were 10-100 times less sensitive than the WTenzymes, consistent with an inhibitory mechanism that involves covalentmodification of Cys436 and Cys22 of RSK2 and NEK2, respectively. Thisnotion was further supported by x-ray co-crystal structures of RSK2bound to two different cyanoacrylamide fragments from Table 2a. Thestructures show unequivocally that Cys436 of RSK2 forms a covalent bondwith the electrophilic beta-carbon of the cyanoacrylamide moiety (seeFIG. 7).

TABLE 2a IC50 values (uM). Entry RSK2 WT RSK2 C436V NEK2 WT NEK2 C22VPLK1 WT 10

5 100 0.90 >10 30 11

<0.75 150 0.109 10 1 12

0.124 40 3 >10 13

0.250 >150 3 >10 14

1 >10 15

0.183 >150 3 >10 16

0.092 17

0.630 >10 18

0.758 0.211 >10 19

20

60 0.2 >10 21

60 1 >10 22

10 150 8 >300 100 23

3 >10 1 24

5 >150 10 25

2.6 6.5 26

27

28

29

1 1 30

>300 >300 >300 31

20 >150 40 >300 300 32

0.142 >10 33

80 >150 10 10 34

0.129 35

0.493

TABLE 2b IC50 values of compounds (μM). RSK2 RSK2 NEK2 NEK2 PLK1 JAK2JAK3 Cmpd WT C436V WT C22V WT WT WT 36

0.440 37

0.747 38

0.386 39

2 40

0.014 0.189 0.650 41

0.014 1.14 0.650 42

0.089 3.8 10 43

0.0038 44

0.0024 45

0.0243 46

0.0232 47

0.0413 48

0.0047 49

0.0065 50

0.0132

TABLE 2c IC50 values of compounds (μM). Cmpd Btk JAK2 JAK3 WT cSrc S345CcSrc 51

0.09 >10 0.273 5.9 0.52 52

0.13 >10 0.141 0.64 0.08 53

1.5 >5.0 2.5 54

0.78 55

0.70 >5.0 0.49 56

0.23 >10 0.660 >5.0 1.2 57

0.62 N/I 2.1 58

0.07 N/I 0.29 59

0.04 60

0.24 61

0.63 N/I N/I

TABLE 2d IC50 values of compounds (μM). Cmpd RSK2 WT 62

0.0105 63

0.071 64

0.0045 65

0.0413 66

0.0036 67

0.0148 68

0.002 69

>0.500 70

>0.500 71

>0.500 72

0.017 73

0.0261 74

>0.500 75

0.0048 76

0.224 77

0.0126 78

0.0304 79

0.274 80

0.0209 81

0.003 82

0.041

TABLE 2e IC50 values of compounds (μM). Cmpd Btk WT cSrc S345C cSrc 83

0.08 >10 >10

Example 72 X-ray Crystallography and Binding Models at the RSK2 CTD ATPSite

Methods. RSK2 CTD was expressed and purified as described above (seedescription for FIG. 1A AND FIG. 1B) and concentrated to 20 mg/ml in 20mM HEPES pH 8.0, 50 mM NaCl. Cyanoacrylamide 6, 12, or 15 (1 μl of a 10mM stock solution in 100% DMSO) was then added to 19 μl of RSK2 CTD, togive 19 mg/ml protein and 0.5 mM inhibitor in 5% DMSO. Crystals of thecomplexes were then grown in hanging drops by mixing 1 μl ofprotein/inhibitor complex with 1 μl of precipitant solution composed of0.1 M HEPES pH 7.0, 10% PEG3350, 50 mM ammonium sulphate at 20° C.Typically, crystals grew to maximal dimensions in 1-2 days. Crystalswere then transferred to a cryoprotectant solution (LV Cryo Oil, MitegenLLC, Ithaca, N.Y.) and frozen in a stream of liquid nitrogen at 100° K.The crystals belonged to the space group P4₁2₁2 with unit cellparameters a=47.5 Å; b=47.5 Å; c=291.5 Å. All datasets were collected onthe 8.2.1 beamline of the Advanced Light Source at the Lawrence BerkeleyNational Laboratory. Diffraction data were integrated and scaled withthe program XDS (Kabsh, W. 1993). The structure of the RSK2CTD/inhibitor complex was solved by molecular replacement using data to2.4 Å (cyanoacrylamide 6), 2.1 Å (cyanoacrylamide 12), and 2.1 Å(cyanoacrylamide 15) and Protein Data Bank structure 2qr8 as a searchmodel in program MolRep (Vagin et al., 1997), followed by several roundsof manual rebuilding and restrained refinement with programs COOT(Emsley P, Cowtan K., Acta Crystallogr D Biol Crystallogr. 2004) andREFMAC5 (GN, Vagin A A, Dodson E J. Acta Crystallogr D Biol Crystallogr.1997).

Cyanoacrylamides, when appended to three different kinase inhibitorscaffolds, bind to the ATP binding pocket of RSK2 CTD and form acovalent bond with Cys-436. We solved co-crystal structures of RSK2 CTDbound to three cyanoacrylamide inhibitors: pyrrolo-pyrimidine 6 (Table1), azaindole 12 (Table 2), and indazole 15 (Table 2). These compoundsare potent inhibitors of RSK2 kinase activity, and mutation of Cys-436to Val confers significant resistance (Table 1 and Table 2). Theco-crystal structures of 6, 12, and 15 reveal non-covalent interactionsthat are typically observed in kinase/inhibitor co-crystal structures;e.g., hydrogen bonding between a heteroatom in the inhibitor and thebackbone NH of RSK2 Met-496. For each of the cyanoacrylamide inhibitors,a covalent bond between the thiol of Cys-436 and the beta-carbon of thecyanoacrylamide moiety is clearly visible (FIG. 7, created from thestructure coordinate files with PyMol). In addition to the datapresented in Table 2, the co-crystal structures shown in FIG. 7 supportthe notion that an acrylonitrile, when substituted on thenitrile-bearing carbon with an electron withdrawing group (e.g.,carboxamide), is a “portable” cysteine-targeting moiety that can beappended to diverse kinase-binding scaffolds to achieve potentinhibitors (nanomolar affinity) that make a reversible covalent bondwith an active site cysteine. Binding of indazole Cmpd 40 to Cys-436 ofRSK2 is depicted in the top panel of FIG. 8.

Example 73 Cloning, Protein Expression, Purification, andCrystallization of cSrc

The kinase domain of chicken cSrc (S345C mutant) was expressed andpurified as described (Blair et al., Nat. Chem. Biol., 2007) andconcentrated to 3 mg/ml in 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT, 5%Glycerol. Protein (3 mg/mL) was incubated for 10 minutes on ice withinhibitor (200 μM) with 2% DMSO. Crystals of the complex were then grownin hanging drops by mixing 1 μl of protein/inhibitor—complex with 1 μlof precipitant solution composed of 100 mM MES, 50 mM NaOAc, 2%PEG(4000), pH 6.5 at 20° C. Typically crystals grew as thin plates tomaximal dimensions in 1-2 days. Crystals were then transferred to acryoprotectant solution consisting of mother liquor supplemented with25% glycerol and frozen in a stream of liquid nitrogen at 100 K.

Data Collection and Structure Solution.

The crystals belonged to the space group P1 with unit cell parametersa=42.0 Å; b=63.2 Å; c=73.1 Å; α=100.9°; β=90.8°; γ=90.0°. All datasetswere collected on the 8.2.1. beamline of the Advanced Light Source atthe Berkeley National Laboratory. Diffraction data were integrated andscaled with the program XDS (Kabsh, W. 1993). The structure of thecSRC/inhibitor complex was solved by molecular replacement using data to2.3 Å and structure 3en4 as search model in program MolRep, followed byseveral rounds of manual rebuilding and restrained refinement withprograms COOT and REFMAC5. Binding of pyrrolopyrimdine Cmpd 55 toCys-345 of cSrc is depicted in the bottom panel of FIG. 8.

Example 74 General Procedure for cSrc Kinase Assay

Wild-type and S345C mutant cSrc kinase domains were expressed andpurified as described (Blair et al., Nat. Chem. Biol., 2007). Thepurified cSrc kinase (2 nM final concentration) was pre-incubated withinhibitors (six or ten concentrations, in duplicate) for 30 minutes atroom temperature in kinase reaction buffer (20 mM HEPES pH 7.4, 10 mMMgCl₂ 0.2 mM EDTA) with 200 μM ATP, and 1 mg/mL BSA. Kinase reactionswere initiated by the addition of 0.05 μCi/μL of γ-³²P-ATP (6000Ci/mmol, NEN) and 0.1 mM substrate peptide (LEIYGEFKKK) (SEQ ID NO:2)and incubated for 30 minutes at room temperature. Kinase activity wasdetermined by spotting 6 μL of each reaction onto sheets ofphosphocellulose. Each blot was washed once with 1% AcOH solution, twicewith 0.1% H₃PO₄ solution, and once with MeOH (5-10 minutes per wash).Dried blots were exposed for 30 minutes to a storage phosphor screen andscanned by a Typhoon imager (GE Life Sciences). The data were quantifiedusing ImageQuant (v. 5.2, Molecular Dynamics) and plotted using GraphPadPrism 4.0 software.

Example 752-cyano-N-(1-hydroxy-2-methylpropan-2-yl)-3-(3-(3,4,5-trimethoxyphenyl)-1H-indazol-5-yl)acrylamide

To a solution of 3-(3,4,5-trimethoxyphenyl)-1H-indazole-5-carbaldehyde(48 mg, 0.16 mmol) and2-cyano-N-(1-hydroxy-2-methylpropan-2-yl)acetamide (23 mg, 0.16 mmol) inTHF (1 mL) was added DBU (16 μL, 0.16 mmol). The colorless solutionslowly became bright orange after 1 hour. The reaction mixtureconcentrated and purified by preparative TLC, eluting with EtOAc, toafford 44 mg (61%) of2-cyano-N-(1-hydroxy-2-methylpropan-2-yl)-3-(3-(3,4,5-trimethoxyphenyl)-1H-indazol-5-yl)acrylamidea bright yellow solid. Exact mass: 450.19, M/z found: 451.23 (M+H)⁺.

Example 76 3-(pyridin-3-yl)-1H-indazole-5-carbaldehyde

3-iodo-5-formylindazole (206 mg, 0.735 mmol), pyridin-3-ylboronic acid(106 mg, 0.882 mmol) and K₂CO₃ (330 mg, 2.21 mmol) were combined in 5:1Dioxane:H₂O (2 mL) and degassed with bubbling argon for 20 minutes.Pd(PPh₃)₄ (91 mg, 0.074 mmol) was added, and the reaction vessel purgedwith argon again. The reaction was then microwaved at 130° C. for 45minutes. The crude reaction mixture was diluted with EtOAc (20 mL) andwashed twice with 1M HCl (20 mL). The combined aqueous washings wereneutralized with 1M NaOH and extracted with three portions of EtOAc (75mL) which were concentrated under reduced pressure to afford 42 mg (26%)of 3-(pyridin-3-yl)-1H-indazole-5-carbaldehyde as a yellow solid.

Example 77 2-cyano-3-(3-(pyridin-3-yl)-1H-indazol-5-yl)acrylamide

To a solution of 3-(pyridin-3-yl)-1H-indazole-5-carbaldehyde (30 mg,0.134 mmol) and 2-cyano-N-methylacetamide (12 mg, 0.134 mmol) in THF (1mL) was added DBU (13 μL, 0.134 mmol). The colorless slurry slowlyturned bright yellow and soluble upon addition of DBU. When all solidshad dissolved, the crude reaction mixture was concentrated and purifiedby preparative TLC, eluting with 95:5:1 EtOAc:MeOH:TEA, to afford 7 mg(18%) of 2-cyano-3-(3-(pyridin-3-yl)-1H-indazol-5-yl)acrylamide as abright yellow solid. Exact mass: 289.10, M/z found: 290.01 (M+H)⁺.

Example 78(E)-3-(3-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)-2-cyano-N-(1-hydroxy-2-methylpropan-2-yl)acrylamide

A 4 mL vial fitted with a magnetic stir bar was charged with3-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzaldehyde(5 mg, 0.01 mmol), 2-cyano-N-(1-hydroxy-2-methylpropan-2-yl)acetamide (5mg, 0.03 mmol), piperidinium acetate (2 mg, 0.02 mmol), and 2-propanol(0.5 mL). The reaction mixture was heated to 60° C. for 24 h. Thesolution was concentrated under reduced pressure and the resultingresidue was purified by Si-gel chromatography (elute 100% EtOAc to 10%MeOH/EtOAc) to yield the product, which was further purified by RP-HPLC(gradient: 20-95% MeCN/water with 0.1% TFA over 25 min) to yield theproduct as a white solid (1.5 mg, 22% yield). Exact Mass: 544.22. M/zfound: 545.0 (M+H)⁺.

Example 794-Amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidine-6-carbaldehyde

To a solution of4-Di-tert-butyloxycarbonylamino-7-(3-tert-butyldimethylsiloxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidine-6-carbaldehyde(1.43 g, 2.28 mmol) in dichloromethane (12 mL) was added TFA (5 mL). Thereaction mixture was maintained at ambient temperature for 12 hours,then concentrated under reduced pressure. The residue was redissolved inTHF (12 mL), and 1M aq. HCl (4 mL) was added. The reaction mixture wasstirred at ambient temperature for 24 hours and then diluted with EtOAc(50 mL) and satd. aq. NaHCO₃ (50 mL). The phases were separated and theaqueous phase was extracted with EtOAc (50 mL). The combined organicextracts were washed with brine (50 mL), then concentrated under reducedpressure. The residue was azeotroped with benzene (50 mL) and dried invacuo to afford 0.99 g of the deprotected aldehyde (wet), which was usedwithout further purification.

Example 80 Synthesis of Cyanoacetamides and Heteroaryl Acetonitriles

3-morpholino-3-oxopropanenitrile,2-cyano-N-(2-(dimethylamino)ethyl)-N-methylacetamide (Wang, K.; Nguyen,K.; Huang, Y.; Dömling, A. J. Comb. Chem. 2009, 11, 920-927) and2-cyano-N-(1,3-dihydroxy-2-methylpropan-2-yl)acetamide (Santilli, A. A.;Osdene, T. S. J. Org. Chem. 1964, 29, 2066-2068) were synthesized aspreviously described.

1. 4-Cyanomethylpyridine-1-oxide

To a slurry of 4-cyanomethylpyridine hydrochloride (689.2 mg, 4.458mmol) in Chloroform (12 mL) was added NaHCO₃ (538 mg, 5.35 mmol, 1.4equiv) in H₂O (2 mL) leading to effervescence. Once gas evolutionsubsided, meta-chloroperbenzoic acid (1.25 g, 6.69 mmol, 1.6 equiv) wasadded in one portion. The reaction mixture was stirred at ambienttemperature for 24 hours, then concentrated with silica gel and purifiedby silica gel chromatography (12.5 to 25% IPA in CH₂Cl₂) afforded 129 mg(22% yield) of the N-oxide as a red semi-solid.

2. 2-Cyano-N-(2-hydroxyethyl)-N-methylacetamide

To a solution of methyl cyanoacetate (6.27 g, 63.3 mmol) in ethanol (12mL) was added 2-(methylamino)ethanol (5.6 mL, 69.6 mmol, 1.1 equiv). Thereaction mixture was heated to 70° C. for 4 hours, then cooled toambient temperature and concentrated under reduced pressure. Silica gelchromatography of the crude residue (EtOAc) afforded 8.76 g (97% yield)of 2-cyano-N-(2-hydroxyethyl)-N-methylacetamide as an amber oil.

3. N-tert-butyl-2-cyanoacetamide

To a slurry of tert-butylamine (3.7 mL, 35.4 mmol, 2.0 equiv) and sodiumcarbonate (3.75 g, 35.4 mmol, 2.0 equiv) in CH₂Cl₂ (20 mL) cooled to 0°C. was added a solution of chloroacetyl chloride (1.4 mL, 17.7 mmol, 1.0equiv) in CH₂Cl₂ (10 mL) over 5 minutes. Once the addition was complete,the reaction mixture was allowed to warm to ambient temperature andstirred for 45 minutes, then filtered. The filter cake was washed withCH₂Cl₂ (50 mL). The combined filtrate and wash were concentrated toafford the intermediate tert-butyl-2-chloroacetamide as white solid,which was carried on to the next step without any further purification.The tert-butyl-2-chloroacetamide was dissolved in DMF (12 mL), and 2.31g (35.4 mmol, 2.0 equiv) of finely crushed KCN was added. The reactionmixture was heated to 70° C. for 2 hours and then filtered. The filtercake was washed with EtOAc (2×30 mL). The combined washes and filtratewere concentrated to afford an amber oil. Purification by silica gelchromatography (40% EtOAc in Hexanes) afforded thetert-butyl-2-cyanoacetamide as a semi-solid with DMF as an impurity.This semi-solid was dissolved in EtOAc (100 mL) and washed with water(3×70 mL). The combined aqueous washes were extracted with EtOAc (100mL). The combined EtOAc solutions were washed with brine (70 mL), dried(MgSO₄) and concentrated to afford the analytically puretert-butyl-2-cyanoacetamide (1.88 g, 76% yield, 2 steps) as an off-whitesolid.

4. N-1-adamantyl-2-cyanoacetamide

To a slurry of 1-adamantylamine (2.80 g, 18.5 mmol, 1.5 equiv) andsodium carbonate (2.62 g, 24.7 mmol, 2.0 equiv) in CH₂Cl₂ (17 mL) at20-25° C. was added a solution of chloroacetyl chloride (1.24 mL, 12.3mmol, 1.0 equiv) in CH₂Cl₂ (5 mL) over 5 minutes. Once the addition wascomplete, the reaction mixture was stirred for 10 minutes and dilutedwith CH₂Cl₂ (10 mL) for more efficient stirring. After 2 hours, thereaction mixture was filtered. The filter cake was washed with CH₂Cl₂(20 mL). The combined filtrate and wash were concentrated to afford theintermediate 1-adamantyl-2-chloroacetamide as white solid, which wascarried on to the next step without any further purification. The1-adamantyl-2-chloroacetamide was dissolved in DMF (6 mL), and 1.61 g(24.7 mmol, 2.0 equiv) of finely crushed KCN was added. The reactionmixture was heated to 70° C. for 14 hours and then cooled to ambienttemperature. The reaction mixture was diluted with EtOAc (100 mL) andwashed with water (3×60 mL). The combined aqueous washes were extractedwith EtOAc (70 mL). The combined EtOAc solutions were washed with brine(100 mL), dried (MgSO₄) and concentrated to afford a residue.Purification by silica gel chromatography (25% EtOAc in Hexanes)afforded 1-adamantyl-2-chloroacetamide (500 mg, 18% yield) and1-adamantyl-2-cyanoacetamide (491 mg, 18% yield, 2 steps) as whitesolids.

5. 1-Cyano-N-isopropylmethanesulfonamide

To a solution of 1-cyanomethanesulfonyl chloride (Sammes, M. P.; Wylie,C. M.; Hoggett, J. G. J. Chem. Soc. (C), 1971, 2151-2155) (3.25 g, 23.3mmol, 1.0 equiv) in THF (20 mL) cooled to 0-5° C. was addedisopropylamine (5.0 mL, 58.2 mmol, 2.5 equiv) over 10 minutes. Thereaction mixture was then allowed to warm to ambient temperature. After12 hours, the reaction mixture was filtered through Celite. The filtercake was washed with MeCN (50 mL). The wash was combined with thefiltrate and concentrated to afford a residue, which after purificationby silica gel chromatography (25% EtOAc in Hexanes), afforded 1.55 g(41% yield) of 1-cyano-N-isopropylmethanesulfonamide as an amber oil.

6. Allyl-2-(2-cyanoacetamido)-2-methylpropanoate

A slurry of 2-amino-2-methylpropanoic acid (Aib, 2.05 g, 19.9 mmol) andp-toluenesulfonic acid monohydrate (5.11 g, 26.9 mmol, 1.35 equiv) inallyl alcohol (30 mL) was heated to 90° C. for 24 hours, then cooled toambient temperature. The reaction mixture was concentrated to removeallyl alcohol and then diluted with CH₂Cl₂ (100 mL), and saturatedaqueous sodium carbonate (100 mL). The phases were separated and theaqueous phase was extracted with CH₂Cl₂ (100 mL). The combined organicextracts were washed with brine (100 mL), dried (Na₂SO₄) andconcentrated to afford the crude Aib-allyl ester (2.84 g, quantitativeyield) as a brown oil, which was carried on to the next step withoutfurther purification.

To a solution of cyanoacetic acid (500 mg, 5.88 mmol, 1.0 equiv),N,N-diisopropylethylamine (3.1 mL, 17.6 mmol, 3.0 equiv) and Aib-allylester (1.26 g, 8.82 mmol, 1.5 equiv) in CH₂Cl₂ (8 mL) cooled to 0-5° C.was added a solution of N,N′-dicyclohexylcarbodiimide (1.82 g, 8.82mmol, 1.5 equiv) in CH₂Cl₂ (7 mL) over 3 minutes. The reaction mixturewas stirred at 0-5° C. for 40 minutes and then allowed to warm toambient temperature. After 24 hours, the reaction mixture was dilutedwith EtOAc (100 mL) and washed with 0.5 M HCl (50 mL) and the organicphase was filtered through Celite to remove precipitated solids. Thefiltrate was washed with brine (50 mL), dried (Na₂SO₄) and concentratedto afford a brown solid, which was purified by silica gel chromatography(50% EtOAc in Hexanes), to afford 648 mg (53% yield) ofallyl-2-(2-cyanoacetamido)-2-methylpropanoate as a white solid.

7. 2-Cyano-N′,N′-dimethylacetohydrazide

To a solution of methyl cyanoacetate (5.48 g, 55.3 mmol, 1.0 equiv) in2-propanol (12 mL) was added N,N-dimethylhydrazine (8.4 mL, 110.6 mmol,2.0 equiv). The reaction mixture was stirred at ambient temperature for20 hours, then concentrated to afford a red solid, which was slurried inEt2O (50 mL), filtered and dried to afford the hydrazide as an orangered solid (4.52 g, 64% yield). Additional purification of a portion ofthe solid by silica gel chromatography (50% EtOAc in Hexanes, thenEtOAc) afforded 1.68 g of analytical pure hydrazide as an off-whitesolid.

8. 2-Cyano-N-methoxyacetamide

To a slurry of methyl cyanoacetate (1.21 g, 12.2 mmol, 1.0 equiv) andO-methylhydroxylamine hydrochloride (2.02 g, 24.2 mmol, 1.9 equiv) in2-propanol (5 mL) was added triethylamine (5.1 mL, 36.3 mmol, 3.0equiv). The reaction mixture was heated to 70° C. for 3 hours and thenfiltered hot. The filter cake was washed with 2-propanol (3×5 mL). Thecombined washes and filtrate were concentrated and the residue affordedwas purified by silica gel chromatography (70% EtOAc in Hexanes) toafford 2-cyano-N-methoxyacetamide (673 mg, 48% yield) as a white solid.

9. 2-(1H-pyrazol-1-yl)acetonitrile

To a slurry of pyrazole (1.04 g, 15.3 mmol, 1.0 equiv) and cesiumcarbonate (7.47 g, 22.9 mmol, 1.5 equiv) in MeCN (21 mL) was addedchloroacetonitrile (1.16 mL, 18.3 mmol, 1.2 equiv) over 3 minutes. Thereaction mixture was stirred at ambient temperature for 2 hours andfiltered. The filter cake was washed with MeCN (2×20 mL). The combinedfiltrate and washes were concentrated and the residue obtained waspurified by silica gel chromatography (25% EtOAc in Hexanes) to afford1.23 g (75% yield) of 2-(1H-pyrazol-1-yl)acetonitrile as a colorlessoil.

10. 2-(1H-1,2,3-triazol-1-yl)acetonitrile

To a slurry of 1H-1,2,3-triazole (261.4 mg, 3.79 mmol, 1.0 equiv) andpotassium carbonate (785 mg, 5.68 mmol, 1.5 equiv) in MeCN (8 mL) wasadded a solution of bromoacetonitrile (303 μL, 4.54 mmol, 1.2 equiv) inMeCN (4 mL) over 3 minutes. The reaction mixture was stirred at ambienttemperature for 2 hours and filtered. The filter cake was washed withMeCN (30 mL). The combined filtrate and washes were concentrated and theresidue obtained was purified by silica gel chromatography (50% EtOAc inHexanes) to afford 211 mg (52% yield) of2-(1H-1,2,3-triazol-1-yl)acetonitrile as a colorless oil.

11. Azetidine (X═H), and 3-hydroxyazetidine (X═OH) cyanoacetamides

Ethylcyanoacetate (1.0 equiv.), azetidine (X═H), or 3-hydroxyazetidine(X═OH) hydrochloride (1.1 equiv.) and triethylamine (1. 5 equiv.) inEtOH (6 mL) were heated at 80° C. for 6 hours. The reaction mixture wasconcentrated and the residue was partitioned between EtOAc (25 mL) andDI water (25 mL). The aqueous phase was extracted with EtOAc (2×30 mL).The combined organic extracts were dried (Na₂SO₄) and concentrated toafford a residue, which was purified by silica gel chromatography (4:1EtOAc/Hexanes for X═H; 16:1 EtOAc/MeOH for X═OH) to afford the desiredcyanoacetamide.

Ethylcyanoacetate (550 mg, 4.86 mmol) and azetidine hydrochloride (500mg) afforded 196.5 mg (33% yield) of azetidine cyanoacetamide.

Ethylcyanoacetate (469.4 mg, 4.15 mmol) and 3-hydroxyazetidinehydrochloride (500 mg) afforded 89 mg (15% yield) of azetidinecyanoacetamide.

Example 81 Synthesis of Cyanoacrylamides or Heteroaryl Acrylonitriles

General procedure for synthesis of cyanoacrylamide orheteroarylacrylonitrile derivatives:4-Amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidine-6-carbaldehyde(1.0 equiv.), the appropriate cyanoacetamide or heteroaryl acetonitrile(1.2-1.5 equiv.) and DBU (1.5-2.0 equiv.) were stirred in THF or DMF (2mL) at ambient temperature for 1-3 days. The reaction mixture was thenconcentrated and purified by preparative TLC or HPLC to afford thecyanoacrylamide or heteroaryl acrylonitrile as a mixture of (E)- and(Z)-isomers.

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-(morpholine-4-carbonyl)acrylonitrile

Yield: 12.1 mg (21% yield). ESI-MS: 447.7 (MH⁺).

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-(pyridin-4-yl)acrylonitrile

Yield: 7.3 mg (25% yield). ESI-MS: 411.7 (MH⁺).

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-N-tert-butyl-2-cyanoacrylamide

Yield: 19.9 mg (38% yield). ESI-MS: 433.2 (MH⁺).

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyano-N-(2-(dimethylamino)ethyl)-N-methylacrylamide

Yield: 9.8 mg (29%). ESI-MS: 462.5 (MH⁺).

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyano-N-(2-hydroxyethyl)-N-methylacrylamide

Yield: 2.3 mg (5%). ESI-MS: 435.6 (MH⁺).

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyano-N-(1,3-dihydroxy-2-methylpropan-2-yl)acrylamide

Yield: 12.3 mg (35% yield). ESI-MS: 464.5 (MH⁺).

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-(111-1,2,4-triazol-1-yl)acrylonitrile

Yield: 7.1 mg (27% yield). ESI-MS: 401.5 (MH⁺).

2-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-1-cyano-N-isopropylethenesulfonamide

Yield: 17.8 mg (49% yield). ESI-MS: 455.5 (MH⁺).

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-N-(1-adamantyl)-2-cyanoacrylamide

Yield: 11.3 mg (27% yield). ESI-MS: 511.6 (MH⁺).

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-(4-methylthiazol-2-yl)acrylonitrile

Yield: 12.7 mg (48% yield). ESI-MS: 431.5 (MH⁺).

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-(1H-pyrazol-1-yl)acrylonitrile

Yield: 6.6 mg (25% yield). ESI-MS: 400.3 (MH⁺).

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-(1H-1,2,3-triazol-1-yl)acrylonitrile

Yield: 5.2 mg (21% yield). ESI-MS: 401.5 (MH⁺).

Allyl-2-(3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyanoacrylamido)-2-methylpropanoate

Yield: 11.1 mg (19% yield). ESI-MS: 503.6 (MH⁺).

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-(pyridin-3-yl)acrylonitrile

Yield: 21.3 mg (62% yield). ESI-MS: 411.3 (MH⁺).

4-(2-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-1-cyanovinyl)pyridine1-oxide

Yield: 3.3 mg (7% yield). ESI-MS: 427.7 (MH⁺).

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyano-N′,N′-dimethylacrylohydrazide

Yield: 6.1 mg (18% yield). ESI-MS: 420.5 (MH⁺).

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyano-N-methoxyacrylamide

Yield: 19 mg (49% yield). ESI-MS: 407.4 (MH⁺).

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyano-N,N-dimethylacrylamide

Prepared from N,N-dimethylcyanoacetamide (Basheer, A.; Yamataka, H.;Ammal, S. C.; Rappoport, Z. J. Org. Chem. 2007, 72, 5297-5312).

Yield: 33.8 mg (24%, E:Z=1.7:1). ESI-MS: 405.2 (MH⁺).

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyano-N-(1-hydroxy-2-methylpropan-2-yl)acrylamide

Prepared from 2-cyano-N-(1-hydroxy-2-methylpropan-2-yl)acetamide(Santilli, A. A.; Osdene, T. S. J. Org. Chem. 1964, 29, 2066-2068).

Yield: 17.2 mg (39%), ESI-MS: 449.2 (MH⁺)

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyano-N,N-diethylacrylamide

Prepared from N,N-diethylcyanoacetamide (Wang, K.; Nguyen, K.; Huang,Y.; Dömling, A. J. Comb. Chem. 2009, 11, 920-927).

Yield: 9.2 mg (8%), ESI-MS: 433.2 (MH⁺)

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-(pyrrolidine-1-carbonyl)acrylonitrile

Prepared from N-(2-cyanoacetyl)pyrrolidine (Wang, K.; Nguyen, K.; Huang,Y.; Dömling, A. J. Comb. Chem. 2009, 11, 920-927).

Yield: 1.6 mg (3%), ESI-MS: 431.2 (MH⁺)

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-(azetidine-1-carbonyl)acrylonitrile

Yield: 16.5 mg (32%), ESI-MS: 417.2 (MH⁺)

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-(3-hydroxyazetidine-1-carbonyl)acrylonitrile

Yield: 20.1 mg (38%), ESI-MS: 433.2 (MH⁺)

(E)-3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-(4-methylpiperazine-1-carbonyl)acrylonitrile

Prepared from N-(2-cyanoacetyl)-N′-methylpiperazine (Proenca, F.; Costa,M. Green Chem. 2008, 10, 995-998).

Yield: 15.7 mg (25%), ESI-MS: 460.2 (MH⁺)

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyano-N-cyclopropylacrylamide

To a solution of4-tert-butyloxycarbonylamino-7-(3-tert-butyldimethylsiloxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidine-6-carbaldehyde(101 mg, 0.1925 mmol) and N-cyclopropylcyanoacetamide³ (47.8 mg, 2.0equiv.) in THF (2.2 mL) that had been pre-cooled to 0-5° C. was addedDBU (58 μL, 2.0 equiv.). The reaction mixture was allowed to warm toambient temperature and stirred for 1 hour, then maintained at −20° C.for 12 hours. The reaction mixture was concentrated and purified bysilica gel chromatography (2:1 Hexanes/EtOAc) to afford 53.2 mg(E:Z=2:1, 44% yield) of the intermediate protected cyanoacrylamide as ayellow oil. This oil was dissolved in CH₂Cl₂ (3 mL) and TFA (1.5 mL) wasadded. After 16 hours at 20-25° C., the reaction mixture wasconcentrated and the residue was redissolved in THF (6 mL) and 1M aq.HCl (2 mL) was added. The reaction mixture was maintained at ambienttemperature for 8 hours, then quenched with satd. aq. NaHCO₃ (20 mL) andbrine (30 mL) and extracted with EtOAc (3×50 mL). The combined EtOAcextracts were dried (MgSO₄) and concentrated and the oil afforded waspurified by preparative TLC (3:1 Toluene/IPA, 0.5 cm plate, 2 elutions)to afford the cyanoacrylamide (26 mg, 74% yield over 2 steps) as ayellow oil. ESI-MS: 417.1 (MH⁺).

1-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2,2-difluoroethanone

To a solution of the corresponding Di-tert-butyloxycarbonylaminotert-butyldimethylsilyloxydifluoromethyl ketone (39.0 mg, 57.8 mmol) inCH₂Cl₂ (2 mL) was added TFA (2 mL). The reaction mixture was maintainedat ambient temperature for 17 hours, then concentrated and the residuewas redissolved in THF (3 mL). HCl (1M, 1 mL) was added and the reactionmixture was stirred at ambient temperature for 12 hours, then quenchedwith saturated aqueous sodium bicarbonate (5 mL) and extracted withEtOAc (3×10 mL). The combined organic extracts were dried (Na₂SO₄) andconcentrated to afford a white solid, which was slurried in MeCN (10mL). The suspension was filtered and the filtrate was concentrated toafford the difluoromethylketone (6.9 mg, 33% yield, 2 steps) as a whitesolid. ESI-MS: 361.3 (MH⁺).

3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyano-N-isopropylpropanamide

To a solution of the corresponding cyanoacrylamide (31.7 mg, 75.8 mmol)in MeOH (1.8 mL) was added glacial AcOH (0.2 mL), followed by sodiumcyanoborohydride (14.3 mg, 227 mmol, 3.0 equiv). The reaction mixturewas stirred at ambient temperature for 24 hours, then concentrated andthe residue purified by preparative HPLC (0.1% TFA in H₂O: 0.1% TFA inMeCN, 95:5 to 20:80 v/v gradient over 20 minutes) to afford the desiredamide (9.8 mg, 24% yield) as a colorless oil. ESI-MS: 421.5 (MH⁺).

Example 82 Assays for Reversible Thiol Addition to Electrophilic,Nitrile-Substituted Olefins

Deuterated Phosphate Buffered Saline (PBS-d) was prepared by dissolutionof NaCl (400 mg), KCl (10 mg), Na₂DPO₄ (30.7 mg) and KD₂PO₄ (9.7 mg) in40 mL D₂O. The pH of the solution was adjusted to 7.4 using NaOD(solution in D₂O) and DCl in D₂O.

To a solution of cyanoacrylate A (5.1 mg, 27.3 μmol) in 0.75 mL ofDMSO-d6 was added a solution of 400 mM 2-mercaptoethanol (BME) in PBS-d(0.25 mL). Analysis of the reaction mixture by ¹H NMR indicated >95%conversion to the thiol adduct B.

A ratio of the starting cyanoacrylate to the adduct can be readilydetermined by integration of the diagnostic peaks at 8.30 ppm for thecyanoacrylate A and 4.52 ppm for the thioether B.

Cyanoacrylate A: ¹H NMR (400 MHz, 3:1 v/v DMSO-d6:PBS-d): δ 8.30 (s,1H), 7.91 (m, 2H), 7.60-7.49 (m, 3H), 3.78 (s, 3H).

Addition of BME causes the formation of adduct B (a ca. 61:39 mixture ofdiastereomers) with the following resonances: ¹H NMR (400 MHz, 3:1 v/vDMSO-d6:PBS-d): 7.41 (m, 1H), 7.35-7.28 (m, 4H), 4.52 (s, 1H), 3.64 (s,3H, major diastereomer), 3.56 (s, 3H, minor diastereomer), 3.46 (t,J=6.3 Hz, 2H, major diastereomer, overlaps with signal for excess BME),3.38 (t, J=6.7 Hz, 2H, minor diastereomer), 2.58-2.40 (m, 2H, overlapswith signal for excess BME). The formation of the thiol adduct was foundto be reversible as demonstrated by the following experiment:

To a solution of cyanoacrylate A (17.1 mg, 91.4 mmol) in 0.75 mL ofDMSO-d6 was added a solution of 400 mM BME in PBS-d (0.25 mL) Analysisof the reaction mixture by ¹H NMR indicated an 85:15 ratio of the thioladduct B to the starting cyanoacrylate A. The reaction mixture was thendiluted 10-fold by addition of 100 μL of the reaction mixture into 900μL of 3:1 v/v DMSO-d6:deuterated PBS. Analysis of the solution by ¹H NMRindicated a 53:47 ratio of the thiol adduct B to the startingcyanoacrylate A; the ratio of cyanoacrylate A to adduct B beingdetermined by the integrals of the diagnostic peaks 8.30 ppm for thecyanoacrylate A and 4.52 ppm for the thioether B (FIG. 9).

Example 83 Thiol Reversibility Assay by UV/Visible Spectroscopy

Reversible reactions of UV-active electrophiles with BME can becharacterized spectrophotometrically using a Spectramax M5 (MolecularDevices, Sunnyvale Calif.) UV-VIS spectrophotometer over the range of250-500 nm at 20° C. Equilibrium reactions were initiated by mixingequal volumes of the N-isopropyl cyanoacrylamide depicted above (400 μMsolution in PBS, pH 7.4) with a solution of 2-mercaptoethanol (100 mMBME solution in PBS, pH 7.4).

Solutions C (200 μM cyanoacrylamide in PBS at pH 7.4, control) and D(200 μM cyanoacrylamide plus 50 mM BME in PBS at pH 7.4) were analyzedspectrophotometrically (Costar clear flat bottom 96-well plate, 100 μLper well for each reaction) by monitoring the cyanoacrylamide absorbancepeak at 400 nm. Disappearance of this peak (solution D, FIG. 2)indicates reaction with BME. Solution D was then diluted 10-fold bymixing 10 μL, aliquots with 90 μL of either PBS (pH 7.4) or 50 mM BME inPBS (pH 7.4). Solution C was also diluted 10-fold in PBS (pH 7.4) andwas used as a reference for the absorbance signal. Dilution of solutionD into PBS results in rapid recovery (within seconds) of thecyanoacrylamide absorbance at 400 nm, indicating rapid reversal of theBME-cyanoacrylamide adduct. These data are consistent with the NMR assaydescribed above. For electrophiles (e.g., cyanoacrylamides) that absorblight at wavelengths above 300 nm, the spectrophotometric assay is moreconvenient than NMR and is more readily implemented in a high-throughputformat (e.g., 96-well plates).

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, databases, Genbank sequences,patents, and patent applications cited herein are hereby incorporated byreference.

1. A method of inhibiting a protein kinase, said method comprisingcontacting said protein kinase with an effective amount of a reversiblekinase inhibitor and allowing said reversible kinase inhibitor toreversibly bind to an active site cysteine residue, thereby inhibitingsaid protein kinase, wherein said reversible kinase inhibitor has thestructure of Formula (I):

wherein R¹ is substituted or unsubstituted heteroaryl; L¹ is a bond,—C(O)—, —C(O)N(L³R²)—, —C(O)O—, —S(O)_(n)—, —O—, —N(L³R²)—,—P(O)(OL³R²)O—, —SO₂N(L³R²)—, —P(O)(NL³R²)N—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; n is 0, 1 or 2; L³ is abond, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene; R² is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; and-L²-E is: —W—X(L⁴R³)_(z)L⁵R⁴ wherein: W is —C(O)— or —S(O)₂—; z is 0 or1; X is O or N, wherein if X is O, then z is 0; R³ and R⁴ areindependently hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl, or R³and R⁴ are joined together with X to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; L⁴ and L⁵are independently a bond, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; or (b) a ring of formula

where ring A is substituted or unsubstituted heteroaryl; wherein saidreversible kinase inhibitor measurably dissociates from said proteinkinase when said protein kinase is not denatured, partially denatured,or fully denatured; and wherein if L¹ is a bond and R¹ is(3-(4-amino-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)propan-1-ol)-6-yl,then -L²-E is not —C(O)NH₂.
 2. The method according to claim 1, whereinsaid reversible kinase inhibitor has the formula:

wherein: W is —C(O)— or —S(O)₂—; z is 0 or 1; X is 0 or N, wherein if Xis 0, then z is 0; R³ and R⁴ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl, wherein R³ and R⁴ are optionally joinedtogether with X to form a substituted or unsubstituted heterocycloalkylor substituted or unsubstituted heteroaryl; R¹ is a substituted 6,5fused ring heteroaryl, a substituted 5,6 fused ring heteroaryl, asubstituted 5,5 fused ring heteroaryl, or a substituted 6,6 fused ringheteroaryl; L², L⁴ and L⁵ are independently is a bond, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; and wherein if L¹ is a bondand R¹ is(3-(4-amino-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)propan-1-ol)-6-yl,then at least one of R³ and R⁴ are is not hydrogen.
 3. (canceled)
 4. Themethod according to claim 1, wherein said reversible kinase inhibitorhas the formula:

wherein the ring A is five-membered R³¹-substituted or unsubstitutedheteroaryl or six-membered R³¹-substituted or unsubstituted heteroaryl;wherein: R³¹ is hydrogen, halogen, —CN, —OH, —COOH, —CF₃,R³³-substituted or unsubstituted alkyl, R³³-substituted or unsubstitutedheteroalkyl, R³³-substituted or unsubstituted cycloalkyl,R³³-substituted or unsubstituted heterocycloalkyl, R³³-substituted orunsubstituted aryl, or R³³-substituted or unsubstituted heteroarylwherein; R³³ is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH,—CF₃, R³⁴-substituted or unsubstituted alkyl, R³⁴-substituted orunsubstituted heteroalkyl, R³⁴-substituted or unsubstituted cycloalkyl,R³⁴-substituted or unsubstituted heterocycloalkyl, R³⁴-substituted orunsubstituted aryl, or R³⁴-substituted or unsubstituted heteroarylwherein; R³⁴ is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH,—CF₃, R³⁵-substituted or unsubstituted alkyl, R³⁵-substituted orunsubstituted heteroalkyl, R³⁵-substituted or unsubstituted cycloalkyl,R³⁵-substituted or unsubstituted heterocycloalkyl, R³⁵-substituted orunsubstituted aryl, or R³⁵-substituted or unsubstituted heteroarylwherein; R³⁵ is independently halogen, —CN, —OH, —NH₂, —COOH, —CF₃,unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, orunsubstituted heteroaryl.
 5. The method according to claim 1, whereinsaid reversible kinase inhibitor has the formula:

wherein ring A is a substituted or unsubstituted heteroaryl. 6.-9.(canceled)
 10. The method according to claim 2, wherein R⁴ is hydrogen,R^(23A)-substituted or unsubstituted alkyl, R^(23A)-substituted orunsubstituted heteroalkyl, R^(23A)-substituted or unsubstitutedcycloalkyl, R^(23A)-substituted or unsubstituted heterocycloalkyl,R^(23A)-substituted or unsubstituted aryl, or R^(23A)-substituted orunsubstituted heteroaryl wherein; R^(23A) is hydrogen, halogen, —CN,—OH, —NH₂, —COOH, —CF₃, R^(24A)-substituted or unsubstituted alkyl,R^(24A)-substituted or unsubstituted heteroalkyl, R^(24A)-substituted orunsubstituted cycloalkyl, R^(24A)-substituted or unsubstitutedheterocycloalkyl, R^(24A)-substituted or unsubstituted aryl,R^(24A)-substituted or unsubstituted heteroaryl, or -L^(7A)-R^(24B)wherein; L^(7A) is independently —O—, —C(O)—, —C(O)NH—, —S(O)_(y′)—, or—S(O)_(y′)NH—; y′ is 0, 1, or 2; R^(24B) is independently hydrogen,halogen, —CN, —OH, —NH₂, —COOH, —CF₃, R^(24A)-substituted orunsubstituted alkyl, R^(24A)-substituted or unsubstituted heteroalkyl,R^(24A)-substituted or unsubstituted cycloalkyl, R^(24A)-substituted orunsubstituted heterocycloalkyl, R^(24A)-substituted or unsubstitutedaryl, R^(24A)-substituted or unsubstituted heteroaryl wherein; R^(24A)is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R^(25A)-substituted or unsubstituted alkyl, R^(25A)-substituted orunsubstituted heteroalkyl, R^(25A)-substituted or unsubstitutedcycloalkyl, R^(25A)-substituted or unsubstituted heterocycloalkyl,R^(25A)-substituted or unsubstituted aryl, or R^(25A)-substituted orunsubstituted heteroaryl wherein; R^(25A) is independently hydrogen,halogen, —CN, —OH, —NH₂, —COOH, —CF₃, R^(26A)-substituted orunsubstituted alkyl, R^(26A)-substituted or unsubstituted heteroalkyl,R^(26A)-substituted or unsubstituted cycloalkyl, R^(26A)-substituted orunsubstituted heterocycloalkyl, R^(26A)-substituted or unsubstitutedaryl, or R^(26A)-substituted or unsubstituted heteroaryl wherein;R^(26A) is independently halogen, —CN, —OH, —NH₂, —COOH, —CF₃,unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, orunsubstituted heteroaryl. 11.-14. (canceled)
 15. The method according toclaim 2, wherein R¹ is R⁷-substituted or unsubstituted heterocycloalkyl,R⁷-substituted or unsubstituted aryl, or R⁷-substituted or unsubstitutedheteroaryl R⁷-substituted 6,5 fused ring heteroaryl, R⁷-substituted 5,6fused ring heteroaryl, R⁷-substituted 5,5 fused ring heteroaryl, orR⁷-substituted 6,6 fused ring heteroaryl; L¹, L⁴ and L⁵ areindependently a bond; R⁷ is independently —NH₂, R⁸-substituted orunsubstituted alkyl, R⁸-substituted or unsubstituted aryl,R⁸-substituted or unsubstituted heteroaryl, or -L⁴-R^(7A) wherein; L⁴ is—C(O)—; R^(7A) is independently R⁸-substituted or unsubstituted alkyl,R⁸-substituted or unsubstituted aryl, R⁸-substituted or unsubstitutedheteroaryl wherein; R⁸ is independently —OH or R⁹-substituted orunsubstituted alkyl; R⁴ is hydrogen or R¹⁵-substituted or unsubstitutedalkyl; R³ is hydrogen or R²³-substituted or unsubstituted alkyl; or R³and R⁴ are optionally joined together with X to form a 4-7 memberedheterocycloalkyl; R⁹ is independently hydrogen, halogen, —CN, —OH,—NH_(z), —COOH, R¹⁰-substituted or unsubstituted alkyl, R¹⁰-substitutedor unsubstituted heteroalkyl, R¹⁰-substituted or unsubstitutedcycloalkyl, R¹⁰-substituted or unsubstituted heterocycloalkyl,R¹⁰-substituted or unsubstituted aryl, or R¹⁰-substituted orunsubstituted heteroaryl; R¹⁰ is independently halogen, —CN, —OH, —NH₇,—COOH, —CF_(a), unsubstituted alkyl, unsubstituted heteroalkyl,unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstitutedaryl, or unsubstituted heteroaryl; R¹⁵ is independently hydro en,halogen, —CN, —OH, —NH₂, —COOH R¹⁶-substituted or unsubstituted alkyl,R¹⁶-substituted or unsubstituted heteroalkyl, R¹⁶-substituted orunsubstituted cycloalkyl, R¹⁶-substituted or unsubstitutedheterocycloalkyl, R¹⁶-substituted or unsubstituted aryl, R¹⁶-substitutedor unsubstituted heteroaryl, or -L⁷-R^(15A) wherein: L′ is —O—, —C(O)—,—C(O)NH—, —S(O)_(Y), or —S(O)_(y)NH—. y is 0, 1, or 2; R^(15A) ishydrogen, halogen, —CN, —OH, —NH₂, —COOH, R¹⁶-substituted orunsubstituted alkyl, R¹⁶-substituted or unsubstituted heteroalkyl,R¹⁶-substituted or unsubstituted cycloalkyl, R¹⁶-substituted orunsubstituted heterocycloalkyl, R¹⁶-substituted or unsubstituted aryl,R¹⁶-substituted or unsubstituted heteroaryl wherein; R¹⁶ isindependently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R¹⁷-substituted or unsubstituted alkyl, R¹⁷-substituted or unsubstitutedheteroalkyl, R¹⁷-substituted or unsubstituted cycloalkyl,R¹⁷-substituted or unsubstituted heterocycloalkyl, R¹⁷-substituted orunsubstituted aryl, or R¹⁷-substituted or unsubstituted heteroarylwherein; R¹⁷ is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH,R¹⁸-substituted or unsubstituted alkyl, R¹⁸-substituted or unsubstitutedheteroalkyl, R¹⁸-substituted or unsubstituted cycloalkyl,R¹⁸-substituted or unsubstituted heterocycloalkyl, R¹⁸-substituted orunsubstituted aryl, or R¹⁸-substituted or unsubstituted heteroarylwherein; R¹⁸ is independently halo en —CN, —OH, —NH₂, —COOH, —CF₃,unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, orunsubstituted heteroaryl; R²³ is independently hydrogen, halogen, —CN,—OH, —NH₂, —COOH, R²⁴-substituted or unsubstituted alkyl,R²⁴-substituted or unsubstituted heteroalkyl, R²⁴-substituted orunsubstituted cycloalkyl, R²⁴-substituted or unsubstitutedheterocycloalkyl, R²⁴-substituted or unsubstituted aryl, R²⁴-substitutedor unsubstituted heteroaryl, or -L⁸-R^(23A′) wherein; L⁸ is —O—, —C(O)—,—C(O)NH—, p is 0, 1, or 2; R^(23A′) is hydrogen halogen —CN, —OH, —NH₂,—COOH, —CF₃, R²⁴-substituted or unsubstituted alkyl, R²⁴-substituted orunsubstituted heteroalkyl, R²⁴-substituted or unsubstituted cycloalkyl,R²⁴-substituted or unsubstituted heterocycloalkyl, R²⁴-substituted orunsubstituted aryl, R²⁴-substituted or unsubstituted heteroaryl wherein;R²⁴ is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH, —CF₃,R²⁵-substituted or unsubstituted alkyl, R²⁵-substituted or unsubstitutedheteroalkyl, R²⁵-substituted or unsubstituted cycloalkyl,R²⁵-substituted or unsubstituted heterocycloalkyl, R²⁵-substituted orunsubstituted aryl, or R²⁵-substituted or unsubstituted heteroarylwherein; R²⁵ is independently hydrogen, halogen, —CN, —OH, —NH₂, —COOH,—CF₃, R²⁶-substituted or unsubstituted alkyl, R²⁶-substituted orunsubstituted heteroalkyl, R²⁶-substituted or unsubstituted cycloalkyl,R²⁶-substituted or unsubstituted heterocycloalkyl, R²⁶-substituted orunsubstituted aryl, or R²⁶-substituted or unsubstituted heteroarylwherein R²⁶ is independently halogen, —CN, —OH, —NH₂, unsubstitutedalkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstitutedheteroaryl.
 16. The method according to claim 15, wherein R⁸ isindependently —OH or unsubstituted alkyl.
 17. The method according toclaim 15, wherein X is N. 18.-22. (canceled)
 23. The method according toclaim 1, wherein said protein kinase is Rsk, Nek, Mekk1, MSK1 or Plk.24. A method of treating a disease associated with kinase activity in asubject in need of such treatment, said method comprising administeringto said subject a therapeutically effective amount of a compound havingthe structure of Formula (I)

wherein R¹ is substituted or unsubstituted heteroaryl; L¹ is a bond,—C(O)N(L³R²)—, —C(O)O—, —S(O)_(n)—, —O—, —S—, —N(L³R²)—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; n is 0, 1 or 2; L³ isindependently a bond, substituted or unsubstituted alkylene, substitutedor unsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene; R² is independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; -L²-E is: (a) —W—X(L⁴R³)_(z)L⁵R⁴ wherein: W is—C(O)— or —S(O)2—; z is 0 or 1; X is O or N, wherein if X is O, then zis 0; R³ and R⁴ are independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl, or R³ and R⁴ are joined together with X to form asubstituted or unsubstituted heterocycloalkyl or substituted orunsubstituted heteroaryl; L⁴ and L⁵ are independently a bond,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene; or (b) a ring offormula

where ring A is substituted or unsubstituted heteroaryl; wherein if L¹is a bond and R¹ is(3-(4-amino-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)propan-1-ol)-6-yl,then at least one of R² and R³ is not hydrogen.
 25. The method accordingto claim 24, wherein said disease or disorder is cancer, autoimmune, HIVinfection or inflammation.
 26. A compound having the formula:

wherein W is —C(O)— or —S(O)₂—; z is 0 or 1; X is O or N, wherein if Xis O, then z is 0; R¹ is a substituted 6,5 fused ring heteroaryl, asubstituted 5,6 fused ring heteroaryl, a substituted 5,5 fused ringheteroaryl, or a substituted 6,6 fused ring heteroaryl; R³ and R⁴ areindependently hydrogen, unsubstituted alkyl, alkyl substituted with oneor two hydroxy or di-(unsubstituted alkylamino, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, or substituted orunsubstituted aryl, wherein R³ and R⁴ are optionally joined togetherwith X to form a substituted or unsubstituted heterocycloalkyl orsubstituted or unsubstituted heteroaryl; L¹, L⁴ and L⁵ are independentlya bond, unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene; and wherein ifL¹ is a bond and R¹ is(3-(4-amino-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)propan-1-ol)-6-yl,then at least one of R³ and R⁴ are not hydrogen.
 27. The compound ofclaim 26 wherein X is N. 28.-34. (canceled)
 35. The compound accordingto claim 27 wherein R¹ is R⁷-substituted 6,5 fused ring heteroaryl, orR⁷-substituted 5,6 fused ring heteroaryl; R⁷ is independently —NH₂,R⁸-substituted or unsubstituted alkyl, R⁸-substituted or unsubstitutedaryl; and R⁸ is independently —OH or unsubstituted alkyl.
 36. Thecompound according to claim 35, wherein R¹ is R⁷-substituted indazolyl,or R⁷-substituted 7H-pyrrolo[2,3-d]pyrimidinyl.
 37. The compoundaccording to claim 36, wherein R³ and R⁴ are hydrogen.
 38. The compoundaccording to claim 36, wherein R³ is unsubstituted alkyl; and R⁴ ishydrogen.
 39. (canceled)
 40. The compound according to claim 36, whereinR³ and R⁴ join with N to form R²³-substituted or unsubstitutedpyrrolidinyl. 41.-43. (canceled)
 44. A compound having the formula:

wherein R¹ is a substituted 6,5 fused ring heteroaryl, a substituted 5,6fused ring heteroaryl, a substituted 5,5 fused ring heteroaryl, or asubstituted 6,6 fused ring heteroaryl; L¹ is a bond, unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; the ring A is a substitutedor unsubstituted heteroaryl.
 45. The compound according to claim 26,having the structure:


46. The compound according to claim 44, having the structure


47. A pharmaceutical composition comprising a compound of any of theclaim 26, 27, 35, 36-38, 40, 44, or 45 or a pharmaceutically acceptableexcipient.